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The development of bionic organ-on-a-chip technology relies heavily on advancements in in situ sensors and biochip packaging. By integrating precise biological and fluid condition sensing with microfluidics and electronic components, long-term dynamic closed-loop culture systems can be achieved. This study aims to develop biocompatible heterogeneous packaging and laser surface modification techniques to enable the encapsulation of electronic components while minimizing their impact on fluid dynamics. Using a kidney-on-a-chip as a case study, a non-toxic packaging process and fluid interface control methods have been successfully developed. Experimentally, miniature pressure sensors and control circuit boards were encapsulated using parylene-C, a biocompatible material, to isolate biochemical fluids from electronic components. Ultraviolet laser processing was employed to fabricate structures on parylene-C. The results demonstrate that through precise control of processing parameters, the wettability of the material can be tuned freely within a contact angle range of 60° to 110°. Morphological observations and MTT assays confirmed that the material and the processing methods do not induce cytotoxicity. This technology will facilitate the packaging of various miniature electronic components and biochips in the future. Furthermore, laser processing enables rapid and precise control of interface conditions across different regions within the chip, demonstrating a high potential for customized mass production of biochips. The proposed innovations provide a solution for in situ sensing in organ-on-a-chip systems and advanced biochip packaging. We believe that the development of this technology is a critical step toward realizing the concept of "organ twin".

The external jugular vein (EJV) has been commonly used for clinical procedures in veterinary medicine, while the internal jugular vein (IJV) has been used mainly for research. Limited literature regarding anatomical variants in both EJV and IJV is available. The objectives of this descriptive retrospective study were to identify the number of dogs with variations of the jugular veins in canine patients undergoing CT of the head and neck and characterize these variations. Of 1000 dogs, 193 dogs (19.3%) had anatomical variations. Six types of anatomical variations were identified. The most common Type I was the absence of the linguofacial vein (72.0%, n = 139), with the lingual, facial, and maxillary veins entering the EJV at a common branching point. Type II described a variable course of the EJV and IJV, which in some dogs resulted in the absence of the EJV (14.0%, n = 27). Type III was a variable anastomotic loop formed by the linguofacial or maxillary vein cranial or at the junction of the EJV (4.2%, n = 8). In Type IV, the EJV was either suspected to be aplastic or markedly reduced in diameter compared with the contralateral side just distal to its formation (3.6%, n = 7). Type V was found in only six dogs (3.1%) with unilateral differences in the diameter of the IJV. Six of 193 dogs had a combination of more than one variation (Type VI, 3.1%). Type I was significantly observed more frequently in meso- and dolichocephalic breeds. Type II was significantly observed more frequently in brachycephalic breeds., (© 2024 American College of Veterinary Radiology.)

Angiostrongylus malaysiensis is a potential zoonotic parasite, which reported to co-occur with A. cantonensis in human cerebrospinal fluid. It is a heteroxenous nematode that primarily develops through the early larval stages in gastropods and attains sexual maturity within rats. This study was conducted to determine the host species responsible for the reservoir of A. malaysiensis and investigate the risk factor for transmission among the hosts in Kuala Lumpur, Malaysia. Sampling was conducted in six recreational parks. The rats were trapped alive using steel wire traps with bait, while the gastropods were collected by active searching. The rats were euthanized and dissected to collect any adult worms observed. The molecular detection of A. malaysiensis was performed by PCR on gastropod tissue samples. Biotic and landscape factors were recorded for risk factor analysis. In total, 82 rats and 330 gastropods were collected throughout the study. Overall, 3.64% of gastropods and 32.9% of rats were infected with A. malaysiensis. Rattus tiomanicus (Malayan wood rat) and Parmarion martensi (Yellow-shelled semi-slug) were found as important hosts for A. malaysiensis. Host species, sampling site and macrohabitat type are risk factors associated with the prevalence of A. malaysiensis infection in rats. For gastropods, host species and sampling site are risk factors that correlate with the parasite detection. In total, 128 adult A. malaysiensis were recovered from the infected rats. The mean intensity of infection with adult A. malaysiensis was 4.65 for Rattus rattus complex and 4.90 for R. tiomanicus. Adult worms were found in the pulmonary artery or right ventricle, while eggs and first-stage larvae were found in capillaries of the caudal lung lobe. Infected lungs showed extravasated red blood cells in the alveolar spaces. The pulmonary arteries in the infected lung lobe were thickened. Kepong Metropolitan Park is the hotspot area for A. malaysiensis in Kuala Lumpur. These results provide essential information for public health officials to develop targeted interventions to reduce the transmission of A. malaysiensis in urban areas, particularly in recreational parks., (© 2023 Wiley-VCH GmbH.)

In Alzheimer's disease (AD), sensome receptor dysfunction impairs microglial danger-associated molecular pattern (DAMP) clearance and exacerbates disease pathology. Although extrinsic signals, including interleukin-33 (IL-33), can restore microglial DAMP clearance, it remains largely unclear how the sensome receptor is regulated and interacts with DAMP during phagocytic clearance. Here, we show that IL-33 induces VCAM1 in microglia, which promotes microglial chemotaxis toward amyloid-beta (Aβ) plaque-associated ApoE, and leads to Aβ clearance. We show that IL-33 stimulates a chemotactic state in microglia, characterized by Aβ-directed migration. Functional screening identified that VCAM1 directs microglial Aβ chemotaxis by sensing Aβ plaque-associated ApoE. Moreover, we found that disrupting VCAM1-ApoE interaction abolishes microglial Aβ chemotaxis, resulting in decreased microglial clearance of Aβ. In patients with AD, higher cerebrospinal fluid levels of soluble VCAM1 were correlated with impaired microglial Aβ chemotaxis. Together, our findings demonstrate that promoting VCAM1-ApoE-dependent microglial functions ameliorates AD pathology., (© 2023. The Author(s).)

Microglia maintain brain homeostasis through their ability to survey and phagocytose danger-associated molecular patterns (DAMPs). In Alzheimer's disease (AD), microglial phagocytic clearance regulates the turnover of neurotoxic DAMPs including amyloid beta (Aβ) and hyperphosphorylated tau. To mediate DAMP clearance, microglia express a repertoire of surface receptors to sense DAMPs; the activation of these receptors subsequently triggers a chemotaxis-to-phagocytosis functional transition in microglia. Therefore, the interaction between microglial receptors and DAMPs plays a critical role in controlling microglial DAMP clearance and AD pathogenesis. However, there is no comprehensive overview on how microglial sensome receptors interact with DAMPs and regulate various microglial functions, including chemotaxis and phagocytosis. In this review, we discuss the important axes of receptor-ligand interaction that control different microglial functions and their roles in AD pathogenesis. First, we summarize how the accumulation and structural changes of DAMPs trigger microglial functional impairment, including impaired DAMP clearance and aberrant synaptic pruning, in AD. Then, we discuss the important receptor-ligand axes that restore microglial DAMP clearance in AD and aging. These findings suggest that targeting microglial chemotaxis-the first critical step of the microglial chemotaxis-to-phagocytosis state transition-can promote microglial DAMP clearance in AD. Thus, our review highlights the importance of microglial chemotaxis in promoting microglial clearance activity in AD. Further detailed investigations are essential to identify the molecular machinery that controls microglial chemotaxis in AD., (© 2023 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

Introduction: Canine leptospirosis has always been a differential diagnosis in dogs presenting with clinical signs and blood profiles associated with kidney and/or liver disease. The conventional polymerase chain reaction (PCR) provides diagnoses, but real-time PCR-based tests provide earlier confirmation and determine the severity of infection, especially in the acute stage, allowing early detection for immediate treatment decisions. To our knowledge, real-time PCR has not been routinely adopted for clinical investigation in Malaysia. This study evaluated TaqMan real-time PCR (qPCR) assays diagnosing leptospirosis and compared their applicability to clinical samples from dogs with kidney and/or liver disease against a conventional PCR reference., Material and Methods: The qPCR assays were validated using existing leptospiral isolates. Whole blood and urine samples were analysed using a conventional PCR, LipL32(1) and LipL32(2) qPCRs and a microscopic agglutination test. The sensitivity and specificity of the qPCRs were determined., Results: The LipL32(1) qPCR assay had more diagnostic value than the LipL32(2) qPCR assay. Further evaluation of this assay revealed that it could detect as low as five DNA copies per reaction with high specificity for the tested leptospiral strains. No cross-amplification was observed with other organisms. Analysing the clinical samples, the LipL32(1) qPCR assay had 100.0% sensitivity and >75.0% specificity., Conclusion: The LipL32(1) qPCR assay is sensitive, specific and has the potential to be applied in future studies., Competing Interests: Conflict of Interests Statement: The authors declare that there is no conflict of interests regarding the publication of this article., (© 2023 Mohammad Sabri Abdul Rahman et al., published by Sciendo.)

The objective of this study was to evaluate the prevalence of involvement of common viral organisms R. equi and K. pneumoniae and their clinicopathological and radiological features in respiratory disease of Malaysian domestic cats. A total of 34 feline cases with acute/chronic infectious respiratory disease signs were followed prospectively to investigate respiratory disease due to R. equi and K. pneumoniae and their relationship with concurrent viral infections in disease manifestation. All sampled cats ( n = 27) were positive for FCoV antibodies and negative for FeLV. A significantly high antibody titer for FCV in n = 26 cases was also noticed. A single sample of pyothorax from a 3-months-old, non-vaccinated kitten was positive for R. equi . Bronchopneumonia with severe infiltration of the polymorphs and mononuclear inflammatory cells were prominent features of lungs histopathology from the kitten positive for R. equi . K. pneumoniae subsp. pneumoniae was confirmed from tracheal swabs of two cats. Histologically, the tracheal tissues of the two cats positive for K. pneumoniae were normal. In diagnostic imaging, epicenter of the infectious URT disease was nasal conchae rostrally and nasal turbinates caudally, however for infectious LRT disease was bronchial tree. Conclusively, infectious respiratory disease is a complex illness in cats, predominantly for unvaccinated kittens and young adult cats, especially those kept in multi-cat household or shelter environments because of the involvement of multiple bacterial and viral organisms as primary or secondary invaders. Clinicians should not preclude feline rhodococcosis from differentials, especially in kittens with pyothorax and less than one year of age. Unlike R. equi , K. pneumoniae has the potential to colonize URT of cats which might be disseminating further to cause LRT disease.

Changes in the levels of circulating proteins are associated with Alzheimer's disease (AD), whereas their pathogenic roles in AD are unclear. Here, we identified soluble ST2 (sST2), a decoy receptor of interleukin-33-ST2 signaling, as a new disease-causing factor in AD. Increased circulating sST2 level is associated with more severe pathological changes in female individuals with AD. Genome-wide association analysis and CRISPR-Cas9 genome editing identified rs1921622 , a genetic variant in an enhancer element of IL1RL1, which downregulates gene and protein levels of sST2. Mendelian randomization analysis using genetic variants, including rs1921622 , demonstrated that decreased sST2 levels lower AD risk and related endophenotypes in females carrying the Apolipoprotein E (APOE)-ε4 genotype; the association is stronger in Chinese than in European-descent populations. Human and mouse transcriptome and immunohistochemical studies showed that rs1921622 /sST2 regulates amyloid-beta (Aβ) pathology through the modulation of microglial activation and Aβ clearance. These findings demonstrate how sST2 level is modulated by a genetic variation and plays a disease-causing role in females with AD., (© 2022. The Author(s).)

Background: The selection of appropriate scaffold plays an important role in ensuring the success of bone regeneration. The use of scaffolds with different materials and their effect on the osteogenic performance of cells is not well studied and this can affect the selection of suitable scaffolds for transplantation. Hence, this study aimed to investigate the comparative ability of two different synthetic scaffolds, mainly hydroxyapatite (HA) and polycaprolactone (PCL) scaffolds in promoting in vitro and in vivo bone regeneration., Method: In vitro cell viability, morphology, and alkaline phosphatase (ALP) activity of MC3T3-E1 cells on HA and PCL scaffolds were determined in comparison to the accepted model outlined for two-dimensional systems. An in vivo study involving the transplantation of MC3T3-E1 cells with scaffolds into an artificial bone defect of 4 mm length and 1.5 mm depth in the rat's left maxilla was conducted. Three-dimensional analysis using micro-computed tomography (micro-CT), hematoxylin and eosin (H&E), and immunohistochemistry analyses evaluation were performed after six weeks of transplantation., Results: MC3T3-E1 cells on the HA scaffold showed the highest cell viability. The cell viability on both scaffolds decreased after 14 days of culture, which reflects the dominant occurrence of osteoblast differentiation. An early sign of osteoblast differentiation can be detected on the PCL scaffold. However, cells on the HA scaffold showed more prominent results with intense mineralized nodules and significantly ( p  < 0.05) high levels of ALP activity with prolonged osteoblast induction. Micro-CT and H&E analyses confirmed the in vitro results with bone formation were significantly ( p  < 0.05) greater in HA scaffold and was supported by IHC analysis which confirmed stronger expression of osteogenic markers ALP and osteocalcin., Conclusion: Different scaffold materials of HA and PCL might have influenced the bone regeneration ability of MC3T3-E1. Regardless, in vitro and in vivo bone regeneration was better in the HA scaffold which indicates its great potential for application in bone regeneration., Competing Interests: The authors declare there are no competing interests., (©2022 Luchman et al.)

Background: Animal models are significant for understanding human osteoarthritis (OA). This study compared the synovial fluid proteomics changes in surgical and chemical induced OA models., Methods: Thirty rabbits either had anterior cruciate ligament transection (ACLT) procedure or injected intra-articularly with monosodium iodoacetate (MIA, 8 mg) into the right knee. The joints were anatomically assessed, and the synovial fluid proteins analyzed using two-dimensional polyacrylamide gel electrophoresis (2DGE) and MALDI TOF/TOF mass spectrometry analysis at 4, 8 and 12 weeks. The proteins' upregulation and downregulation were compared with control healthy knees., Results: Seven proteins (histidine-rich glycoprotein, beta-actin-like protein 2 isoform X1, retinol-binding protein-4, alpha-1-antiproteinase, gelsolin isoform, serotransferrin, immunoglobulin kappa-b4 chain-C-region) were significantly expressed by the surgical induction. They characterized cellular process (27%), organization of cellular components or biogenesis (27%), localization (27%) and biological regulation (18%), which related to synovitis, increased cellularity, and subsequently cartilage damage. Three proteins (apolipoprotein I-IV precursor, serpin peptidase inhibitor and haptoglobin precursor) were significantly modified by the chemical induction. They characterized stimulus responses (23%), immune responses (15%), biological regulations (15%), metabolism (15%), organization of cellular components or biogenesis (8%), cellular process (8%), biological adhesions (8%) and localization (8%), which related to chondrocytes glycolysis/death, neovascularization, subchondral bone necrosis/collapse and inflammation., Conclusions: The surgical induced OA model showed a wider range of protein changes, which were most upregulated at week 12. The biological process proteins expressions showed the chemical induced joints had slower OA progression compared to surgical induced joints. The chemical induced OA joints showed early inflammatory changes, which later decreased., Competing Interests: The authors declare there are no competing interests., (©2022 Syed Sulaiman et al.)

Canine leptospirosis is commonly associated with kidney and/or liver disease. It has been widely reported and causes public health concerns due to its zoonotic potential and its re-emergence, resulting from close contact between humans and dogs. The current study identified potential risk and predictive factors for dogs diagnosed with kidney and/or liver disease due to leptospirosis. A total of 124 client-owned dogs were recruited, and information such as signalment, medical history, management, and clinical findings were documented. Samples collected from the recruited dogs were directly tested using polymerase chain reaction (PCR) and subsequently inoculated for bacterial isolation. Statistical analyses were descriptively analyzed, and risk analyses were performed using Pearson chi-square tests and logistic regression. A total of 53 dogs (42.7%) were positive for leptospiral infection based on PCR, and 10 leptospiral isolates were successfully recovered from eight dogs. The mortality rate of infected dogs was 34.0% (18/53). Medium and large dog breeds, with a history of exposure to rats, and managed outdoors had a greater risk for leptospirosis ( p < 0.05). The significant predictors for the dogs' positivity were the presence of rats and acute clinical illness ( p < 0.05). Administration of antibiotics and the detection of clinical illness at an early stage of the disease improved the survivability of the dogs ( p < 0.05). Identifying the profile of dogs that are at risk to leptospirosis could be useful in the design of diagnostic and treatment strategies, as well as to increase awareness for prevention of the disease.

Alzheimer's disease (AD), the most common neurodegenerative disease, has limited treatment options. As such, extensive studies have been conducted to identify novel therapeutic approaches. We previously reported that rhynchophylline (Rhy), a small molecule EphA4 inhibitor, rescues impaired hippocampal synaptic plasticity and cognitive dysfunctions in APP/PS1 mice, an AD transgenic mouse model. To assess whether Rhy can be developed as an alternative treatment for AD, it is important to examine its pharmacokinetics and effects on other disease-associated pathologies. Here, we show that Rhy ameliorates amyloid plaque burden and reduces inflammation in APP/PS1 mice. Transcriptome analysis revealed that Rhy regulates various molecular pathways in APP/PS1 mouse brains associated with amyloid metabolism and inflammation, specifically the ubiquitin proteasome system, angiogenesis, and microglial functional states. These results show that Rhy, which is blood-brain barrier permeable, is beneficial to amyloid pathology and regulates multiple molecular pathways.

Background: Osteoarthritis (OA) is a multifaceted condition that affects both the subchondral bones and the articular cartilage. Animal models are widely used as an effective supplement and simulation for human OA studies in investigating disease mechanisms and pathophysiology. This study is aimed to evaluate the temporal changes of bone and cartilage in surgically and chemically induced osteoarthritis using micro-computed tomography and histology., Methods: Thirty rabbits underwent either anterior cruciate ligament transection (ACLT) procedure or injected intraarticularly with monosodium iodoacetate (MIA, 8 mg) at the right knee joint. The subchondral bones were scanned via micro-CT, and articular cartilage was assessed histologically at 4-, 8- and 12-week post-induction., Results: Based on bone micro-architecture parameters, the surgically induced group revealed bone remodelling processes, indicated by increase bone volume, thickening of trabeculae, reduced trabecular separation and reduced porosity. On the other hand, the chemically induced group showed active bone resorption processes depicted by decrease bone volume, thinning of trabeculae, increased separation of trabecular and increased porosity consistently until week 12. Histologically, the chemically induced group showed more severe articular cartilage damage compared to the surgically induced group., Conclusions: It can be concluded that in the ACLT group, subchondral bone remodelling precedes articular cartilage damage and vice versa in the MIA group. The findings revealed distinct pathogenic pathways for both induction methods, providing insight into tailored therapeutic strategies, as well as disease progression and treatment outcomes monitoring., (© 2021. The Author(s).)

Leptospirosis is an endemic zoonoses of global proportions. Stray dogs have been postulated to play a role in disease transmission; however, supporting information are still limited. Roaming behavior may not only predispose the dogs to infection, but could also contribute to disease spread. In this study, the susceptibility of urban stray dogs in shedding Leptospira spp. was determined. Blood, urine, and tissue samples of kidney and liver were collected from 100 dogs from 2 animal control facilities. Serological testing using microscopic agglutination test (MAT) were performed on blood against 20 leptospiral serovars with a cut-off titre of ≥ 1:100. Samples were cultured onto semi-solid Ellinghausen and McCullough modified by Johnson and Harris (EMJH) media. Isolates were identified using molecular polymerase chain reaction (PCR) using 2 primers (16s rRNA and LipL32) and hyperimmune serum (HIS) MAT. The seroprevalence for the dogs positive for leptospirosis was 32% (n=32/100) with the following detected serovars: Javanica (n=13), Bataviae (n=10), Icterohaemorrhagiae (n=3), Autumnalis (n=2), Canicola (n=1), Pyrogenes (n=1), Copenhageni (n=1), and Australis (n=1). Six Leptospira spp. isolated were procured from urine (n=2), kidney (n=2) and liver (n=2). All 6 isolates belonged to L. interrogans, a pathogenic variant of Leptospira spp. Serotyping and phylogenetic analysis suggested serovar Bataviae (n=5) and serovar Canicola (n=1). Presence of vaccinal serovars (Icterohaemorrhagiae and Canicola) suggested potential post-vaccination antibodies but the predominance of non-vaccinal serovars (Javanica and Bataviae) indicate the possibility of current infection or post-exposure. Isolation of Leptospira spp. directly from urine sample not only suggested an active infection but highlighted the potential shedding capability among these stray dogs. These findings further strengthen speculations that urban stray dogs could play a role in transmission and dissemination of leptospirosis through their constant movement. The urine of infected dogs may contaminate the environment, posing a major public health threat., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Leptospirosis is a serious bacterial disease that affects both humans and animals. A wide range of symptoms have been described in humans; the disease in dogs is commonly associated with kidney and/or liver disease. In Malaysia, information about the common serovars infecting dogs is limited. Therefore, we investigated the occurrences of leptospirosis in 124 pet dogs diagnosed with kidney and/or liver disease. Blood, urine, abdominal effusion, and/or kidney and liver were collected from the dogs. Based on microscopic agglutination testing, 53 of 124 (42.7%) dogs were seropositive for leptospiral exposure. Sera were frequently positive to serovars Bataviae ( n  = 12), Javanica ( n  = 10), and Icterohaemorrhagiae ( n  = 10). Direct detection using PCR showed that 42 of 124 (33.9%) of the whole blood and 36 of 113 (31.9%) urine samples were positive for pathogenic Leptospira spp. By PCR, 2 of 23 (9.1%) kidney and 2 of 23 (9.1%) liver were positive for pathogenic Leptospira spp. Abdominal effusion from 4 dogs were PCR-positive for pathogenic Leptospira spp. The species detected were L. interrogans , L. borgpetersenii , L. kirschneri , and L. kmetyi by partial 16S rRNA sequencing. We further identified and characterized 11 Leptospira spp. isolates from 8 dogs as serovars Bataviae, Javanica, and Australis. The mortality rate of the Leptospira -infected dogs was high (18 of 53; 34%).

This retrospective study aimed to determine the etiological, clinicopathological, and radiographic features and outcome of feline pyothorax cases. Medical records from twenty-eight cats with pyothorax aged from 4 months to 10 years (median 10 months) diagnosed between 2013 and 2020 were reviewed. Dyspnoea (75.0%), abnormal lung sounds (75.0%) and open-mouth breathing (64.3%) were the predominant respiratory signs. Leucocytosis (61.5%), particularly monocytosis (68.0%), and hyperglobulinaemia (65.4%) were among the most prominent findings in blood analysis. Bilateral pleural effusion was found in 67.9% of the thoracic radiographs. A total of 47.4% of the cytological samples revealed the presence of bacteria, while all had positive bacterial growth. Pasteurella multocida , E. coli , Streptococcus spp., and Staphylococcus spp. were the predominant aerobic bacteria isolated from pleural effusion samples. A chest tube was placed in 64.3% of the cats and 66.7% of cats with chest tubes survived. In total, 46.4% of cats with pyothorax recovered. Amoxicillin-clavulanate was the antimicrobial of choice against aerobic bacteria found in this study and should be given in combination with antimicrobials that cover anaerobic bacteria. Chest tube placement is crucial for treatment success. Cytological results and bacterial culture may not be consistent; thus, bacterial culture should be performed for every case.

Genetic analyses have revealed the pivotal contribution of microglial dysfunctions to the pathogenesis of Alzheimer's disease (AD). Along AD progression, the accumulation of danger-associated molecular patterns (DAMPs) including beta-amyloid and hyperphosphorylated tau continuously stimulates microglia, which results in their chronic activation. Chronically activated microglia secrete excessive pro-inflammatory cytokines, which further regulate microglial responses towards DAMPs. This has spurred longstanding interest in targeting cytokine-induced microglial responses for AD therapeutic development. However, the cytokine-induced microglial state transition is not comprehensively understood. Cytokines are assumed to induce microglial state transition from a resting state to an activated state. However, recent evidence indicate that this microglial state transition involves multiple sequential functional states. Moreover, the mechanisms by which different functional states within the cytokine-induced microglial state transition regulate AD pathology remain unclear. In this review, we summarize how different cytokine signaling pathways, including those of IL-33 (interleukin-33), NLRP3 inflammasome-IL-1β, IL-10, and IL-12/IL-23, regulate microglial functions in AD. Furthermore, we discuss how the modulation of these cytokine signaling pathways can result in beneficial outcomes in AD. Finally, we describe a stepwise functional state transition of microglia induced by cytokine signaling that can provide insights into the molecular basis of the beneficial effects of cytokine modulation in AD and potentially aid therapeutic development.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder with no disease-modifying treatment. AD progression is characterized by cognitive decline, neuroinflammation, and accumulation of amyloid-beta (Aβ) and neurofibrillary tangles in the brain, leading to neuronal and glial dysfunctions. Neuropeptides govern diverse pathophysiological processes and represent key players in AD pathogenesis, regulating synaptic plasticity, glial cell functions and amyloid pathology. Activation of the pro-opiomelanocortin (POMC)-derived neuropeptide and its receptor from the melanocortin receptor (MCR) family have previously been shown to rescue the impairment in hippocampus-dependent synaptic plasticity in the APP/PS1 mouse model of AD. However, the functional roles of MCR signaling in AD conditions, particularly in glial functions, are largely unknown. In this study, we investigated the potential benefits of MCR activation in AD. In APP/PS1 transgenic mice, we demonstrate that MCR activation mediated by the central administration of its agonist D-Tyr MTII substantially reduces Aβ accumulation, while alleviating global inflammation and astrocytic activation, particularly in the hippocampus. MCR activation prominently reduces the A1 subtype of reactive astrocytes, which is considered a key source of astrocytic neurotoxicity in AD. Concordantly, MCR activation suppresses microglial activation, while enhancing their association with amyloid plaques. The blunted activation of microglia may contribute to the reduction in the neurotoxic phenotypes of astrocytes. Importantly, transcriptome analysis reveals that MCR activation restores the impaired homeostatic processes and microglial reactivity in the hippocampus in APP/PS1 mice. Collectively, our findings demonstrate the potential of MCR signaling as therapeutic target for AD.

Alzheimer's disease is characterized by the deposition of extracellular amyloid-beta (Aβ) plaques. While microglial phagocytosis is a major mechanism through which Aβ is cleared, there is no method for quantitatively assessing Aβ phagocytic capacity of microglia in vivo . Here, we present a flow cytometry-based method for investigating the Aβ phagocytic capacity of microglia in vivo . This method enables the direct comparison of Aβ phagocytic capacity between different microglial subpopulations as well as the direct isolation of Aβ phagocytic microglia for downstream applications. For complete details on the use and execution of this protocol, please refer to Lau et al. (2020)., Competing Interests: The authors declare no competing interests., (© 2020 The Authors.)

Ficus deltoidea has been shown to possess antioxidant properties that could prevent the development of chronic inflammatory bone diseases. In this study, the efficacy of F. deltoidea in preventing alveolar bone resorption in osteoporotic rats induced by ovariectomy (OVX) was investigated. Twenty-four female Wistar rats were divided into four groups ( n  = 6) consisting of sham-operated (SO), ovariectomized control (OVXN), ovariectomized treated with estrogen (OVXP), and ovariectomized treated with F. deltoidea extract (OVXF). At the beginning of the study, two nonovariectomized, healthy rats were sacrificed to serve as baseline (BL). Treatment of the rats commenced two weeks after ovariectomy-the OVXP rats that served as positive control received Premarin® (64.5  μ g/kg body weight), while OVXF rats were given F. deltoidea (800 mg/kg body weight); both agents were administered orally for two months. The negative control group of rats (OVXN) and the SO group received deionized water, also administered via oral gavage. At necropsy, morphometric assessment of the interradicular bone of the first molar was carried out using a micro-CT scanner, while quantification of osteoclasts and osteoblasts was performed histologically. The results showed that no statistically significant differences among the groups ( p > 0.05) for bone morphometric assessment. However, trabecular thickness in the OVXF group was similar to BL, while trabecular separation and alveolar bone loss height were lower than those of the OVXN group. Histologically, the OVXF group demonstrated a significantly lower number of osteoclasts and a higher number of osteoblasts compared with OVXN ( p =0.008 and p =0.019, respectively; p < 0.05). In conclusion, F. deltoidea has the capacity to prevent alveolar bone loss in ovariectomy-induced osteoporosis rats by potentially preserving trabecular bone microarchitecture and to decrease osteoclast and increase osteoblast cell count., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this paper., (Copyright © 2020 N. I. Omar et al.)

Background: Osteoporotic-osteoarthritis is an incapacitating musculoskeletal illness of the aged., Objectives: The anti-inflammatory and anti-catabolic actions of Diclofenac were compared with apigenin-C-glycosides rich Clinacanthus nutans (CN) leaf extract in osteoporotic-osteoarthritis rats., Methods: Female Sprague Dawley rats were randomized into five groups (n = 6). Four groups were bilateral ovariectomised for osteoporosis development, and osteoarthritis were induced by intra-articular injection of monosodium iodoacetate (MIA) into the right knee joints. The Sham group was sham-operated, received saline injection and deionized drinking water. The treatment groups were orally given 200 or 400 mg extract/kg body weight or 5 mg diclofenac /kg body weight daily for 28 days. Articular cartilage and bone changes were monitored by gross and histological structures, micro-CT analysis, serum protein biomarkers, and mRNA expressions for inflammation and catabolic protease genes., Results: HPLC analysis confirmed that apigenin-C-glycosides (shaftoside, vitexin, and isovitexin) were the major compounds in the extract. The extract significantly and dose-dependently reduced cartilage erosion, bone loss, cartilage catabolic changes, serum osteoporotic-osteoarthritis biomarkers (procollagen-type-II-N-terminal-propeptide PIINP; procollagen-type-I-N-terminal-propeptide PINP; osteocalcin), inflammation (IL-1β) and mRNA expressions for nuclear-factor-kappa-beta NF-κβ, interleukin-1-beta IL-1β, cyclooxygenase-2; and matrix-metalloproteinase-13 MMP13 activities, in osteoporotic-osteoarthritis rats comparable to Diclofenac., Conclusion: This study demonstrates that apigenin-C-glycosides at 400 mg CN extract/kg (about 0.2 mg apigenin-equivalent/kg) is comparable to diclofenac in suppressing inflammation and catabolic proteases for osteoporotic-osteoarthritis prevention. Graphical abstract.

The incidence of leptospirosis seems to be on the rise and could be an alarming indirect indication of a global re-emergence. It is a potential public health threat when dogs are speculated to be involved in the transmission of leptospirosis through possible subclinical harbouring of Leptospira spp. and subsequent shedding into the environment. This study aimed to detect anti-leptospiral antibodies among dogs and their handlers using the microscopic agglutination test (MAT). Blood samples from 266 apparently healthy dogs and 194 dog handlers were collected at four working dog organisations and four dog shelters. Serum samples were tested using MAT against 20 leptospiral serovars with a cut-off titre >=1:100 (dog) and >=1:50 (dog handlers). Seventy dogs (70/266; 26.3%) were seropositive mainly against serovars Icterohaemorrhagiae, Ballum, Bataviae and Javanica (titres ranged: 1:100-1:800). Sixty-seven dog handlers (67/194; 34.5%) were seropositive mainly against serovars Grippotyphosa, Icterohaemorrhagiae and Malaysia (titres ranged: 1:50-1:200). Dogs were seropositive due to exposure, vaccination or active infection. Seropositive dog handlers could indicate exposure or active infection. This shows the potential of dogs in maintaining and spreading the infection in Malaysia. Due to the occupational risk as a result of frequent contact with dogs and exposure to contaminated environments, dog handlers should be made aware of the presence of this zoonotic disease.

Alzheimer's disease (AD) is the most common form of dementia but has no effective treatment. A comprehensive investigation of cell type-specific responses and cellular heterogeneity in AD is required to provide precise molecular and cellular targets for therapeutic development. Accordingly, we perform single-nucleus transcriptome analysis of 169,496 nuclei from the prefrontal cortical samples of AD patients and normal control (NC) subjects. Differential analysis shows that the cell type-specific transcriptomic changes in AD are associated with the disruption of biological processes including angiogenesis, immune activation, synaptic signaling, and myelination. Subcluster analysis reveals that compared to NC brains, AD brains contain fewer neuroprotective astrocytes and oligodendrocytes. Importantly, our findings show that a subpopulation of angiogenic endothelial cells is induced in the brain in patients with AD. These angiogenic endothelial cells exhibit increased expression of angiogenic growth factors and their receptors (i.e., EGFL7 , FLT1 , and VWF ) and antigen-presentation machinery (i.e., B2M and HLA-E ). This suggests that these endothelial cells contribute to angiogenesis and immune response in AD pathogenesis. Thus, our comprehensive molecular profiling of brain samples from patients with AD reveals previously unknown molecular changes as well as cellular targets that potentially underlie the functional dysregulation of endothelial cells, astrocytes, and oligodendrocytes in AD, providing important insights for therapeutic development., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Background: This paper describes the result of workshops conducted to increase the knowledge and awareness of university students using a multidisciplinary, collaborative, multisectoral and trans-disciplinary approach concerning One Health and the indigenous people of peninsular Malaysia called the Orang Asli ., Methods: A non-experimental pre and post-test intervention study was carried out among medical, veterinary and allied health students from six public and private universities who attended workshops on One Heath in two Orang Asli communities living by the Temenggor lake in Malaysia as part of the Malaysia One Health University Network (MYOHUN) efforts in training future and present One Health workforce., Results: There was a significant increase in various aspects of knowledge and interest concerning One Health and the Orang Asli . The mean knowledge scores of One Health (p < 0.001) and Orang Asli (p < 0.001) increased significantly post workshop. A repeated measures ANOVA with a Greenhouse-Geisser correction showed the mean scores of knowledge of One Health F (1, 166) = 127.198, p < 0.001) and Orang Asli F (1, 166) = 214.757, p < 0.001) differed statistically significantly between the two time points. The test revealed that the score differences for knowledge on One Health (mean difference = 1.796, p < 0.001) and Orang Asli (mean difference = 4.940, p < 0.001) were statistically significant. Repeated measures ANOVA showed a significant difference in the knowledge on Orang Asli between the students of different courses F (4,166) = 3.734, p-0.006. The difference in the One Health knowledge scores between the students of different courses was not statistically significant F (4,166) = 0.998, p = 0.410., Conclusions: Emphasis on field training in relation to One health can provide university students greater levels of preparedness to combat zoonotic diseases., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2020.)

Objectives: This retrospective study aimed to describe clinical manifestations, diagnostic options, radiological features, therapeutic plans and outcomes for cats infected with Rhodococcus equi ., Methods: Forty cats aged between 2 months and 11 years old (median 6 months) that were definitively diagnosed with rhodococcosis between 2012 and 2018 were recruited in this study. Medical records were reviewed for information on signalment, history, clinical presentation, diagnostic testing, treatment plans and clinical outcomes., Results: Of the 40 cats, 36 showed the pulmonary form of the disease, with 35 (87.5%) presenting with dyspnoea, while four cats presented with only cutaneous lesions. Mean body temperature was 38.7 ± 0.2 ° C. Dyspnoea was noted in 87.5% of the cats. Leukocytosis (58.3%) with band neutrophilia (83.3%), monocytosis (58.3%) and thrombocytopenia (55.5%) were prominent findings in the haematology reports. Hyperproteinaemia (61.1%) with hypoalbuminaemia (22.2%) and hyperglobulinaemia (63.8%) with a low albumin:globulin ratio (38.9%) were prominent features of blood biochemistry reports. An alveolar-interstitial pattern was noted in 75% of pre-thoracocentesis radiographs. Pleural effusion, hepatomegaly, thoracic lymphadenopathy and atelectasis of any lung lobe were seen in 88.9%, 75%, 41.7% and 36.1% of cats, respectively. Overall, the mortality rate was 67.5% in both forms., Conclusions and Relevance: Clinicians should be aware that feline rhodococcosis manifests as a pulmonary disease at a much higher rate than previously reported. Further studies are required to address the epidemiology, pathophysiology, disease management and prognosis of feline rhodococcosis. The role of immunosuppression as a predisposing factor in feline rhodococcosis requires further investigation.

Diabetic cataract causes severe vision loss. This study evaluated the effects of hesperidin-standardized Citrus hystrix leaf flavonoids-rich extract (CLE) on diabetic-cataract development. Streptozotocin-induced diabetic rats were orally given 150 and 300 mg CLE/kg body-weight. These were compared with non-treated diabetic or healthy rats as controls, over 8 weeks. The CLE gradually attenuated fasting blood glucose (FBG), biomarkers for inflammation (Tumor necrosis factor alpha TNF-α; prostaglandin E2 PGE2); vascular permeability, (Vascular endothelial growth factor VEGF); and oxidative stress, (malondialdehyde MDA). The diabetic cataract was significantly mitigated by the 150 mg CLE/kg dose. Good correlations were found between cataract incidence with FBG (r 2  = 0.90), serum PGE2 (r 2  = 0.91), MDA (r 2  = 0.99), VEGF (r 2  = 0.71), but not with TNF-α levels (r 2  = 0.49) suggesting the serum FBG, PGE2, MDA, and possibly the VEGF levels may help to predict the cataract risks. The CLE mitigated cataract probably by attenuating hyperglycaemia, inflammation, lens fluid influx, vascular leakage, lens osmotic-imbalance, and fibers over-hydration. PRACTICAL APPLICATIONS: The study shows the flavonoids-rich Citrus hystrix leaf consumption, effectively attenuated diabetes (fasting blood glucose) and mitigated diabetic cataract. It help reduce diabetes-related hyperglycaemia, oxidative stress, inflammation, and vascular leakage. The evidences were the CLE consumptions reduced the serum biomarkers tumor necrosis factor-alpha TNF-α; prostaglandin E2 PGE2, vascular endothelial growth factor (VEGF), and malondialdehyde (MDA). The C. hystrix leaf contains hesperidin, apiin, diosmin, saponarin, apigetrin, rutin and xanthotoxol, and other flavonoid glucosides. The study also showed good correlations between cataract incidence with fasting blood glucose FBG (r 2  = 0.90), serum PGE2 (r 2  = 0.91), and MDA (r 2  = 0.99), and less closely with VEGF (r 2  = 0.71) suggesting these serum biomarkers may help predict cataract risks. The CLE indicated cataract mitigation properties probably by attenuating FBG, inflammation, lens fluid influx, lens osmotic-imbalance, and fibers over-hydration., (© 2020 Wiley Periodicals LLC.)

Impairment of microglial clearance activity contributes to beta-amyloid (Aβ) pathology in Alzheimer's disease (AD). While the transcriptome profile of microglia directs microglial functions, how the microglial transcriptome can be regulated to alleviate AD pathology is largely unknown. Here, we show that injection of interleukin (IL)-33 in an AD transgenic mouse model ameliorates Aβ pathology by reprogramming microglial epigenetic and transcriptomic profiles to induce a microglial subpopulation with enhanced phagocytic activity. These IL-33-responsive microglia (IL-33RMs) express a distinct transcriptome signature that is highlighted by increased major histocompatibility complex class II genes and restored homeostatic signature genes. IL-33-induced remodeling of chromatin accessibility and PU.1 transcription factor binding at the signature genes of IL-33RM control their transcriptome reprogramming. Specifically, disrupting PU.1-DNA interaction abolishes the microglial state transition and Aβ clearance that is induced by IL-33. Thus, we define a PU.1-dependent transcriptional pathway that drives the IL-33-induced functional state transition of microglia, resulting in enhanced Aβ clearance., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Leptospirosis is one of the most widespread zoonotic diseases and can infect both humans and animals worldwide. Healthy cat, as a potential source of exposure to humans, are likely underestimated owing to the lack of overt clinical signs associated with Leptospira spp. infection in this species. The aim of the study was to determine the exposure, shedding, and carrier status of leptospires in shelter cats in Malaysia by using serological, molecular, and bacteriological methods. For this study, 82 healthy cats from two shelters were sampled. The blood, urine, and kidneys were tested using the microscopic agglutination test (MAT), polymerase chain reaction (PCR), and bacterial culture. On the basis of serological, molecular, and/or culture techniques, the total detection of leptospiral infection was 29.3% (n = 24/82). Through culture techniques, 16.7% (n = 4/24) of the cats that tested positive were carriers with positive kidney cultures, and one cat was culture positive for both urine and kidney. The Leptospira spp. isolates were identified as pathogenic L. interrogans serovar Bataviae through serological and molecular methods. Through serological techniques, 87.5% (n = 21/24) had positive antibody titers (100-1600) and most of the Bataviae serogroup (n = 19/21). Using PCR, 16.7% (n = 4/24) of cats were shown to have pathogenic Leptospira spp. DNA in their urine. Furthermore, three out of four culture positive cats were serology negative. The present study reports the first retrieval of pathogenic leptospires from urine and kidneys obtained from naturally infected cats. The results provide evidence of the potential role of naturally infected cats in the transmission of leptospires. Additionally, leptospiral infection occurs sub-clinically in cats. The culture isolation provides evidence that healthy cats could be reservoirs of leptospiral infection, and this information may promote the development of disease prevention strategies for the cat population., Competing Interests: The authors have declared that no competing interests exist.

Chondrosenescence (chondrocyte senescence) and subchondral bone deterioration in osteoarthritic rats were analyzed after treatment with the estrogenic herb Labisia pumila (LP) or diclofenac. Osteoarthritis (OA) was induced in bilaterally ovariectomized (OVX) rats by injecting mono-iodoacetate into the right knee joints. Rats were grouped (n = 8) into nontreated OVX+OA control, OVX+OA + diclofenac (5 mg/kg) (positive control), OVX+OA + LP leaf extract (150 and 300 mg/kg) and healthy sham control. After 8 weeks' treatment, their conditions were evaluated via serum biomarkers, knee joint histology, bone histomorphometry, protein and mRNA expressions. The LP significantly reduced cartilage erosion, femur bone surface alteration, bone loss and porosity and increased trabecular bone thickness better than diclofenac and the non-treated OA. The cartilage catabolic markers' (matrix metalloproteinase (MMP)-13, RUNX2, COL10a, ERa, CASP3 and HIF-2 alpha) mRNA expressions were down-regulated and serum bone formation marker, PINP, was increased by LP in a dose-dependent manner. The LP (containing myricetin and gallic acid) showed protection against chondrosenescence, chondrocyte death, hypoxia-induced cartilage catabolism and subchondral bone deterioration. The bone and cartilage protective effects were by suppressing proteases (collagen break-down), bone resorption and upregulating subchondral bone restoration. The cartilage ER alpha over-expression showed a strong positive correlation with MMP-13, COL10 alpha1, histological, micro-computed tomography evidence for cartilage degradation and chondrosenescence.

Adult hippocampal neurogenesis involves the lifelong generation of neurons. The process depends on the homeostasis of the production of neurons and maintenance of the adult neural stem cell (NSC) pool. Here, we report that α2-chimaerin, a Rho GTPase-activating protein, is essential for NSC homeostasis in adult hippocampal neurogenesis. Conditional deletion of α2-chimaerin in adult NSCs resulted in the premature differentiation of NSCs into intermediate progenitor cells (IPCs), which ultimately depleted the NSC pool and impaired neuron generation. Single-cell RNA sequencing and pseudotime analyses revealed that α2-chimaerin-conditional knockout (α2-CKO) mice lacked a unique NSC subpopulation, termed Klotho-expressing NSCs, during the transition of NSCs to IPCs. Furthermore, α2-CKO led to defects in hippocampal synaptic plasticity and anxiety/depression-like behaviors in mice. Our findings collectively demonstrate that α2-chimaerin plays an essential role in adult hippocampal NSC homeostasis to maintain proper brain function., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Vernonia amygdalina (VA) is a medicinal tropical herb for diabetes and malaria and believed to be beneficial for joint pains. The antiosteorthritis effects of VA leaf in cartilage explant assays and on postmenopausal osteoarthritis (OA) rat model were investigated. The VA reduced the proteoglycan and nitric oxide release from the cartilage explants with interleukin 1β (IL-1β) stimulation. For the preclinical investigation, ovariectomized (OVX) female rats were grouped (n = 8) into nontreated OA, OA + diclofenac (5 mg/kg), OA + VA extract (150 and 300 mg/kg), and healthy sham control. Monosodium iodoacetate was injected into the knee joints to accelerate OA development. After 8 weeks, the macroscopic, microscopic, and histological images showed that the OA rats treated with VA 300 mg/kg and diclofenac had significantly reduced cartilage erosions and osteophytes unlike the control OA rats. The extract significantly down-regulated the inflammatory prostaglandin E2, nuclear factor κβ, IL-1β, ADAMTS-5, collagen type 10α1, and caspase3 in the OVX-OA rats. It up-regulated the anti-inflammatory IL-10 and collagen type 2α1 mRNA expressions, besides reducing serum collagenases (MMP-3 and MMP-13) and collagen type II degradation biomarker (CTX-II) levels in these rats. The VA (containing various caffeoyl-quinic acids, flavanone-O-rutinoside, luteolin, apigenin derivative and vernonioside D) suppressed inflammation, pain, collagenases as well as cartilage degradation, and improved cartilage matrix synthesis to prevent OA., (© 2019 John Wiley & Sons, Ltd.)

This study determined the potential risk factors that may contribute to seropositivity among dogs and dog handlers from working dog and dog shelter institutions. Data was collected from dogs ( n = 266) and dog handlers ( n = 161) using a standardised guided questionnaire. Serum obtained from the dogs and dog handlers was tested using the microscopic agglutination test (MAT). A logistic regression analysis was used to predict leptospiral seropositivity of dogs and dog handlers based on potential risk factors. A total of 22.2% of dogs and 21.7% of dog handlers were seropositive. The significant predictors for the dogs' seropositivity were presence of rats (OR = 4.61 (95% CI: 1.05, 20.33), p = 0.043) and shared common area (OR = 5.12 (95% CI: 1.94, 13.46), p = 0.001) within the organisation. Significant predictor for dog handler seropositivity was contact time with the dogs of more than six hours/day (OR = 3.28 (95% CI: 1.28, 8.40), p = 0.013) after controlling for the effect of other risk factors such as small mammal contact, rat infestation at home, flooding at housing area (within three months) and urban locality. The exposure to various disease sources identified poses risk to dogs and dog handlers. Risk could be reduced with adequate application of protection at work while handling dogs and thus limiting contact with these sources and reducing exposure to infection.

The scopoletin (coumarin) and epicatechin (flavonoid) rich Morinda citrifolia L. (MC) Noni leaves are non-toxic (unlike the fruits) and consumed as vegetables. The anti-osteoarthritis effects of the MC leaf extract against joint cartilage degradation and inflammation were investigated through cartilage explant cultures and pre-clinical animal study. Osteoarthritis were induced by intra-articular monosodium iodoacetate injection into the right knee. The extract, scopoletin and epicatechin, suppressed glycosaminoglycan and nitric oxide release from the cartilage explant in the presence of Interleukin-1β. After 28 days, the extract treatment reduced the in vivo serum levels and joint tissues mRNA expressions for joint cartilage degradation, aggrecanase, and collagenase biomarkers. The extract increased the bone formation marker PINP levels, besides improving the articular cartilage structure and chondrocytes cellularity. The extract improved bone formation/repair, subchondral bone structure, strength and integrity, as well as cartilage synthesis by suppressing inflammation, nitric oxide production, joint catabolism by proteases, and oxidative stress. PRACTICAL APPLICATIONS: The scopoletin (coumarin) and epicatechin (flavonoid) rich Morinda citrifolia (Noni) leaves may be used as vegetables, functional food ingredient, or dietary supplements to suppress osteoarthritis progression against joint cartilage degradation and inflammation. The extract, scopoletin, or epicatechin, suppressed glycosaminoglycan, and nitric oxide release from the cartilage. The Morinda citrifolia leaf extract suppressed inflammation, nitric oxide production, tissues catabolism by proteases and oxidative stress to help reduce joint cartilage degradation, besides improving the articular cartilage structure, chondrocytes health, subchondral bone structure, bone formation/repair, and cartilage synthesis., (© 2019 Wiley Periodicals, Inc.)

Leptospirosis is one of the most widespread zoonotic diseases and despite extensive research, there is still a paucity of information regarding this disease in cats. The aim of this study was to investigate the prevalence of leptospirosis among the shelter cat population in Malaysia and to determine the most common infective Leptospira serogroups among them. Blood samples were collected from a total of 110 cats from 4 different shelters. The sampled cats appeared healthy, with minimal evidence of feline upper respiratory disease. The Microscopic Agglutination Test was used to detect anti-Leptospira antibodies against 20 pathogenic serovars. Based on a cut-off antibody titer of ≥1:100, 20 of 110 sheltered cats, showed presence of anti-Leptospira antibodies against at least 1 serovar. The serodetection of leptospirosis was 18.18% (95% confidence interval 12.09-26.42). The most commonly detected serogroups were Bataviae, Javanica, and Ballum, with antibody titers ranging from 1:100 to 1:1600. Knowledge of the predominant infective serovars in hosts worldwide and regionally is imperative for understanding the epidemiology of this zoonotic disease. Serosurveillance is the first step in this process. Further studies are warranted for investigation of urinary shedding in naturally infected cats with leptospirosis, using Polymerase chain reaction (PCR) and organism isolation followed by serovars identification., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The tropical herb Labisia pumila is traditionally used in facilitating childbirth and post-partum care. The effects of L. pumila leaf extract (LP) in explant cartilage culture and on postmenopausal osteoarthritis (OA) rat model were assessed. The LP (10, 25 and 50 µg/ml) or diclofenac (10 µg/ml) was added to the cartilage explants containing bovine IL-1β (20 ng/ml), to evaluate their direct effects on cartilage degradation. In the preclinical study, rats were grouped (n = 8) into: non-treated OA; OA + diclofenac (5 mg/kg); OA + LP extract (150 and 300 mg/kg); and healthy control. To induce OA, monosodium iodoacetate was injected into the ovariectomised female rats' intra-articular knee joints and evaluated for OA severity after 8 weeks via physical (radiological, macroscopic and histological observations), biochemical, ELISA and mRNA expression analysis (for inflammation and cartilage degradation biomarkers). The LP reduced the nitric oxide and proteoglycan release from the cartilage explants under IL-1β induction. The radiological, macroscopic, microscopic and histological images showed the OA rats treated with LP and diclofenac had significantly reduced osteophytes and cartilage erosions compared to non-treated OA rats. The extract significantly up-regulated the anti-inflammatory interleukin-10, collagen type II and down-regulated pro-inflammatory PTGS2 (prostaglandin-endoperoxide synthase 2) mRNA expressions compared to non-treated control. The LP treatment significantly reduced serum collagenases (MMP-1 and MMP-3) and collagen type II degradation biomarker (CTX-II) levels in OA rats. The LP containing myricetin and gallic acid suppressed inflammation, collagenases and cartilage degradation, and helped cartilage matrix synthesis, to prevent OA at the dose equivalent to 30-60 mg/kg daily for humans.

Osteoporosis (OP) and osteoarthritis (OA) are debilitating musculoskeletal diseases of the elderly. Ficus deltoidea (FD) or mistletoe fig, a medicinal plant, was pre-clinically evaluated against OP- and OA-related bone alterations, in postmenopausal OA rat model. Thirty twelfth-week-old female rats were divided into groups (n = 6). Four groups were bilateral ovariectomized (OVX) and OA-induced by intra-articular monosodium iodoacetate (MIA) injection into the right knee joints. The Sham control and OVX-OA non-treated groups were given deionized water. The three other OVX-OA groups were orally administered daily with FD extract (200, 400 mg/kg) or diclofenac (5 mg/kg) for 4 weeks. The rats' bones and blood were evaluated for protein and mRNA expressions of osteoporosis and inflammatory indicators, and micro-CT computed tomography for bone microstructure. The non-treated OVX-OA rats developed severe OP bone loss and bone microstructural damage in the subchondral and metaphyseal regions, supported by reduced serum bone formation markers (osteocalcin, osteoprotegerin) and increased bone resorption markers (RANKL and CTX-I). The FD extract significantly (p < 0.05) mitigated these bone microstructural and biomarker changes by dose-dependently down-regulating pro-inflammatory NF-κβ, TNF-α, and IL-6 mRNA expressions. The FD extract demonstrated good anti-osteoporotic properties in this OP/OA preclinical model by stimulating bone formation and suppressing bone resorption via anti-inflammatory pathways. This is among the few reports relating the subchondral bone plate and trabecular thickening with the metaphyseal trabecular osteopenic bone loss under osteoporotic-osteoarthritis conditions, providing some insights on the debated inverse relationship between osteoporosis and osteoarthritis.

The effect of Orthosiphon stamineus aqueous (OSA) extract against osteoarthritis (OA) was investigated in explant cartilage culture and in postmenopausal OA rat model. Female rats were bilaterally ovariectomized (OVX). Osteoarthritis was induced after surgical recovery, by intra-articular injection of monosodium iodoacetate (MIA) into the right knee. Rats were grouped (n = 8) into: healthy sham control; non-treated OA; OA + diclofenac (positive control 5 mg/kg); and two doses OSA (150-300 mg/kg). After 4 weeks' treatment, rats were evaluated for OA-related parameters and biomarkers. The OSA reduced proteoglycan and ROS release from the cartilage explants under inflammatory (IL-1b) conditions. In the OA-induced rats' cartilages, the OSA downregulated the mRNA expressions for IL-1β, IL-6, IL-10, TNF-α, NF-κβ, NOS2, PTGS2, PTGER2, ACAN, COL2A1, MMP1, MMP13, ADAMTS4, ADAMTS5 and TIMP1, mostly dose-dependently. The OSA reduced the OA rats' serum levels for PGE 2 , CTX-II, TNF-α, MMP1, MMP13, PIINP, OPG, RANKL, OC and BALP, but not dose-dependently. The OSA contained polyphenols and flavonoids (tetramethoxyflavone). The OSA alleviated articular cartilage degradation, inflammation, collagenase/aggrecanase activities, to improve joint and subchondral bone structure. O. stamineus mitigated osteoarthritis by downregulating inflammation, peptidases and aggrecanases, at a dose equivalent to about 30 mg/kg for humans.

Objective: This study aimed to explore the usefulness of adjunctive buccal and palatal corticotomy for adult maxillary expansion in an animal model using cone-beam computed tomography (CBCT)., Methods: Twelve adult sheep were randomly divided into two groups (each n = 6): a control group, where no treatment was administered, and a treatment group, where buccal and palatal corticotomy-assisted maxillary expansion was performed. CBCT scans were taken before (T1) and after (T2) treatment. Differences in all transverse dental and alveolar dimensions, alveolar width at crest level, hard palate level, horizontal bone loss, interdental cusp width and inter-root apex were assessed using Wilcoxon signed-rank and Mann-Whitney U -tests. Kruskal-Wallis tests and pairwise comparisons were used to detect the significance of differences among the inter-premolar and inter-molar widths., Results: CBCT data revealed significant changes in all transverse dental and alveolar dimensions. The mean interpremolar alveolar width showed an increase of 2.29 to 3.62 mm at the hard palate level, 3.89 to 4.38 mm at the alveolar crest level, and 9.17 to 10.42 mm at the buccal cusp level. Dental changes in the vertical dimension were not significant., Conclusions: Our findings based on an adult animal model suggest that adjunctive buccal and palatal corticotomy can allow for both skeletal and dental expansion, with the amount of dental expansion exceeding that of skeletal expansion at alveolar crest and hard palate levels by two and three folds, respectively. Therefore, this treatment modality is potential to enhance the outcomes of maxillary expansion in adults., Competing Interests: The authors report no commercial, proprietary, or financial interest in the products or companies described in this article.

Objective: Orthosiphon stamineus (OS) or Misai Kucing (Java tea) is a popular herbal supplement from Southeast Asia for various metabolic, age-related diseases. This study investigated the potential use of OS leaf extracts to ameliorate osteoporosis in ovariectomized rats., Methods: Fifty-six female Sprague-Dawley rats were randomly allocated into eight groups (n = 7): SHAM (healthy sham control); OVX (ovarietomized) nontreated rats (negative control); OVX + Remifemin (100 mg/kg body weight), and 2% green tea extract (positive controls); OVX + OS 50% ethanolic and aqueous extracts, both at either 150 or 300 mg/kg. After 16 weeks, the rats' bones and blood were evaluated for osteoporosis indicators (protein and mRNA expressions), micro-computed tomography for bone histomorphometry, and three-point bending test for tibia mechanical strength., Results: The extracts dose-dependently and significantly (P < 0.05) improved bone strength and flexibility, bone mineral density, bone formation protein markers (P1NP), and bone histomorphometry. All extracts reduced the inflammation biomarker (interleukin-6). The extracts up-regulated osteoblastogenesis (bone morphogenetic protein-2) and collagen-1 synthesis (collagen type 1 alpha-1) mRNA expressions, and down-regulated bone resorption (TNFSF11 and nuclear factor-kappa B) mRNA expressions. Both the water and 50% ethanolic extract were effective. The effective dose is equivalent to 25 to 50 mg/kg extract for humans., Conclusions: The extract showed bone-protective and antiosteoporotic effects (improving bone strength, flexibility, bone density, and bone morphometry) by reducing inflammation and the bone resorption biomarkers, while enhancing bone formation biomarkers and collagen synthesis.

The effect of scopoletin-standardized Morinda elliptica leaf extract against osteoarthritis was investigated in ex vivo explant culture and preclinical rodent model. Thirty male rats were grouped (n = 6) into untreated osteoarthritis (OA), OA + Diclofenac (5 mg/kg), and OA + extract (200 and 400 mg/kg) and compared with healthy control. Monosodium iodoacetate were injected into the right intra-articular knee joints to induce OA. The rats were evaluated for OA severity via physical (micro-CT and histological observations), biochemical, ELISA, and mRNA expression analysis (for inflammation and cartilage degradation biomarkers), after 28 days of treatment. The extract suppressed glycosaminoglycan release from the cartilage explant in the presence of Interleukin-1β. The 200 mg/kg dose appeared better than 400 mg/kg dose, at reducing cartilage and subchondral bone erosions in OA-induced rats, by significantly down-regulating the collagenases and aggrecanase. The extract dose-dependently reduced serum inflammation biomarkers and increased bone formation biomarkers to near normal levels in the OA-induced rats. M. elliptica leaf scopoletin-standardised extract alleviated OA progression and articular cartilage structure, by ameliorating cartilage degradation, nitric oxide levels, inflammation, bone /cartilage homeostasis, collagenase/aggrecanase activities, chondrocytes survival, subchondral bone structure and integrity., (Copyright © 2017 John Wiley & Sons, Ltd.)

Working dogs are canine animals that have been trained to assist human beings in carrying out various tasks. They help in guarding property, performing rescues, assisting the visually impaired or physically handicapped, searching for drugs, explosives, and others. Leptospirosis is one of the most widespread zoonotic diseases in the world and a commonly occurring disease of the tropics and subtropics. In Malaysia, all working dogs are normally vaccinated with serovars, Pomona, Icterohaemorrhagiae, Canicola, and Grippotyphosa based on protocols recommended from other countries. The duration of immunity in vaccinated dogs for Leptospira can last up to 13 months; however, there is no full crossprotection between the different serovars. Five representative canine units from different government agencies in Malaysia (n = 96 dogs) were recruited in this study. For detection, the microscopic agglutination test was performed by incubating the serum from dogs with various serovars of leptospires, namely, Icterohaemorrhagiae, Canicola, Pomona, Grippotyphosa, Australis, Bataviae, Javanica, Tarassovi, Hebdomadis, Lai, and Pyrogenes. The plasma obtained was used for polymerase chain reaction (PCR) analysis, for the detection of 16S rRNA, and lipL 32 genes of Leptospira. Out of the 96 dogs sampled, only 3 dogs were positive toward serovars, Australis, Bataviae, and Javanica, based on the cutoff point at 1:80. The seroprevalence of canine leptospirosis in this population was 3.1% (n = 3/96). However, all 96 blood samples of working dogs tested negative for both pathogenic and nonpathogenic Leptospira genes. The results revealed that, by vaccination alone, working dogs were not fully protected against leptospirosis and could pose a risk to dog handlers. A preventative and control protocol for leptospirosis is warranted, and its implementation should be monitored and improved accordingly from time to time, in order to maintain a healthy condition in both working dogs and their handlers., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objective: Ficus deltoidea Jack (mistletoe fig) is an ornamental plant found in various parts of the world and used as traditional herbal medicine in some countries. This study investigated the potential use of F deltoidea leaf extract to mitigate osteoarthritis (OA) in ovariectomized (estrogen-deficient postmenopausal model) rats and the mechanisms involved. Diclofenac was used for comparison., Methods: Sprague-Dawley female rats (12 weeks old) were divided randomly into five groups (n = 6): healthy; nontreated OA; OA + diclofenac (5 mg/kg); OA + extract (200 mg/kg); and OA + extract (400 mg/kg). Two weeks after bilaterally ovariectomy, OA was induced by intra-articular injection of monosodium iodoacetate into the right knee joints. After 28 days of treatment, the rats were evaluated for knee OA via physical (radiological and histological observations), biochemical, enzyme-linked immunosorbent assay, and gene expression analysis, for inflammation and cartilage degradation biomarkers., Results: The osteoarthritic rats treated with the extract, and diclofenac showed significant reduction of cartilage erosion (via radiological, macroscopic, and histological images) compared with untreated osteoarthritic rats. The elevated serum interleukin-1β, prostaglandin E2, and C-telopeptide type II collagen levels in osteoarthritic rats were significantly reduced by F deltoidea leaf extract comparable to diclofenac. The extract significantly down-regulated the interleukin-1β, prostaglandin E2 receptor, and matrix metalloproteinase-1 mRNA expressions in the osteoarthritic cartilages, similar to diclofenac., Conclusions: F deltoidea leaf extract mitigated postmenopausal osteoarthritic joint destruction by inhibiting inflammation and cartilage degradation enzymes, at an effective extract dose equivalent to about 60 mg/kg for humans. The main bioactive compounds are probably the antioxidative flavonoids vitexin and isovitexin.

This study investigated canine vector borne diseases (CVBDs) of zoonotic significance in three major shelters in Peninsular Malaysia in order to highlight the importance of shelter management in diseases prevention. Since crowded housing has the potential to bring vectors and infected dogs into close proximity, the prevalence of CVBDs infection is always higher in sheltered dogs in comparison to pet dogs. Serum was collected from 90 adult dogs (30 from each shelter) from three shelters in different regions of Peninsular Malaysia (Shelter A, B and C). The prevalence of Dirofilaria immitis, Ehrlichia canis, E. ewingii, Anaplasma phagocytophilum, A. platys and Borrelia burgdorferi in dogs residing in three shelters with different management protocols was investigated by using rapid clinical test kits, SNAP® 4Dx® Plus (IDEXX Laboratories, Westbrook, ME). The most common pathogen detected was Ehrlichia spp. (55.6%, 50 out of 90 dogs), followed by Anaplasma spp. (16.7%; 15 out of 90 dogs). Dirofilaria immitis could only be detected in one of the shelters (10.0%; nine out of 90 dogs). No evidence of B. burgdorferi was detected in all three shelters. Forty one out of 90 dogs (45.6%) were infected with at least one pathogen meanwhile 14 out of 90 dogs (15.6%) were infected with more than one pathogen. It is of crucial importance to practice preventive measures during the admission of the new animals into the facilities. Occasional treatment of the dogs (as practiced in Shelter B) was not effective when compared to scheduled preventive measures. Future studies should emphasize the isolation and additional molecular characterization of vector borne pathogens in Peninsular Malaysia. In addition, studies that define the tick species that infest dogs in this region would help to elucidate their role as vectors for disease transmission.

This case study is to report the proteins detected by proteomic analysis of synovial fluid from a dog diagnosed with idiopathic immune-mediated polyarthritis, and to compare it with healthy dogs. Synovial fluid was collected via arthrocentesis from a dog diagnosed with immune-mediated polyarthritis. Protein precipitation was performed on the synovial fluid, followed by isoelectric focusing and 2-dimensional gel electrophoresis. The spots on the 2-dimensional gels were analyzed using MALDI-TOF/MS. The results were then analyzed against the MASCOT database. The results from the proteomic analysis revealed an abundance of several types of immunoglobulins together with the presence of complement C4b-binding protein alpha chain. Actin and keratin were also among the proteins detected. Proteomic studies, facilitate a better understanding of the different levels of proteins expressed during disease activity. Potential disease biomarkers can aid in the diagnosis of disease, as well as help in monitoring treatment efficacy and providing prognosis for the patient., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Leptospirosis is under-reported and remarkably neglected in Malaysia, especially in companion animals. In recent years, dogs have become popular pets and potentially act as one of the risk factors for human leptospiral infection. The purpose of this study was to determine the serological and molecular status of leptospirosis in healthy and dogs with kidney disease in Klang Valley, Malaysia and to gain insight of the possible serovars involved in the dog population in Klang Valley, Malaysia. Blood samples were obtained from 57 dogs (19 kidney disease patient; 38 healthy dogs, respectively). Serum samples obtained from these animals were screened for leptospiral antibodies by the microscopic agglutination test (MAT). Polymerase chain reaction (PCR) assay was performed on plasma samples to detect leptospiral DNA. By MAT, three out of 19 (15.8%) dogs with kidney disease were positive for L. canicola. One out of 38 (2.6%) healthy dogs was positive for L. icterohemorrhagiae. The overall seroprevalence for leptospirosis in dogs in Klang Valley, Malaysia was 7.0% (n=4/57). Only one out of the 19 dogs (5.3%) with kidney disease was tested positive to pathogenic Leptospira by PCR assay. All the 38 healthy dogs were negative. Positive results in healthy dogs and dogs with kidney disease for leptospirosis warrant further investigation of leptospirosis in dog population in Malaysia. The prevalence and incidence of this disease in the dog population in this country need further investigation.

A study on seroprevalence and molecular detection of canine leptospirosis was carried out in a dog population (randomly selected n=80 dogs) from an animal shelter X. All the dogs in Shelter X appeared clinically healthy. Eighty blood samples were obtained and their serum were serologically examined using Microscopic Agglutination Test (MAT) against 10 Leptospira serovars. Plasma samples obtained were subjected to Polymerase Chain Reaction (PCR) assay. Three out of 80 dogs (3.8%) tested positive for L. bataviae based on MAT at a titer of 1:80. The seroprevalence of 9 other Leptospira serovars was not evident in this study. All the dogs tested negative against leptospirosis with PCR assay. In conclusion, canine leptospirosis was detected in dogs in this animal shelter. L. bataviae was identified as the infecting serovar. To our knowledge, this is the second report of serovar Bataviae infection in dogs in Malaysia. The 3 dogs in our study could possibly be a source of leptospiral infection to other dogs and may shed the bacteria into the environment. This serovar is not available in canine vaccination programs, therefore the dogs are not protected from this disease. Further investigation is warranted to determine whether the infected dogs are carriers of this serovar.

Objective: To describe the radiographic, computed tomographic (CT), and arthroscopic findings in different age groups of Labrador Retrievers diagnosed with medial coronoid disease (MCD), and to compare the ulnar subtrochlear sclerosis (STS) observed on radiographs with the ratio between the mean attenuation of the ulnar subtrochlear bone and the mean attenuation of the cortical bone measured on CT., Study Design: Prospective clinical study., Animals: Dogs (n = 31; 31 elbow joints) and 6 healthy Labrador Retrievers (6 elbow joints)., Methods: Radiographic, CT, and intraoperative arthroscopic images (2008-2012) were evaluated. Statistical analysis was performed for the descriptive study to evaluate the difference in findings between age groups and to investigate the correlation between radiographic and CT evaluated ulnar STS., Results: Ulnar STS (87.6%) was the most common radiographic findings in dogs ≤12 months and blurring of the cranial edge of the medial coronoid process (MCP; 66.7%) was the most common radiographic findings in dogs >12 months. MCP fragmentation was the most common CT finding in both age groups (93.8% [≤12 months]; 66.7% [>12 months]). A displaced fragment (68.8%) was the most common arthroscopic finding in dogs ≤12 months whereas osteochondromalacia (53.3%) was the most common finding in dogs >12 months. Sensitivity of radiography in detecting MCD was 93.8% (≤12 months) and 73.3% (>12 months) and for CT was 93.8% (≤12 months) and 66.7% (>12 months). Radiographic evaluated ulnar STS was strongly correlated with CT evaluated ulnar STS., Conclusion: Wide ranges of radiographic, CT, and arthroscopic findings in Labrador Retrievers diagnosed with medial coronoid disease were identified., (© Copyright 2014 by The American College of Veterinary Surgeons.)

Left atrial thrombi are a significant cause of cardioembolic morbidity and mortality. Transesophageal echocardiography is the preferred method for complete visualization of atrial thrombi, and has a sensitivity and specificity of up to 100% and 99%, respectively. Cardiac magnetic resonance imaging has been shown to be useful in identifying tissue characteristics that may aid in differentiating between atrial myxoma and thrombi. This is an unusual case of a large, free-floating atrial thrombus with a cystic appearance that was surgically removed. The echocardiographic appearance of the cystic atrial mass led to the consideration of another potential etiology for a cardiac mass, namely, atrial myxoma. Histopathologic analyses of the mass led to the final diagnosis of an atrial thrombus.

Objectives: To compare the development, monitored by radiography and computed tomography, of the antebrachia and elbow joints in seven Labrador Retrievers with healthy elbow joints and in seven Labrador Retrievers that developed medial coronoid disease (MCD), in order to determine whether disturbances in the development of the antebrachia and elbow joints, between the age of six and 17 weeks may lead to medial coronoid disease., Methods: A prospective study of 14 Labrador Retrievers in their active growth stage was performed. The development of the antebrachia and elbow joints was assessed between six and 17 weeks of age using radiography and computed tomography determining the development of secondary ossification centres, radioulnar length ratio, radial angulation, and inter-relationship between the humerus, ulna and radius., Results: For the parameters of ossification of secondary ossification centres, radioulnar length ratio, radial angulation, and joint congruence evaluation, there was no significant difference in the development of the antebrachia and elbow joints of seven Labrador Retrievers positive and seven Labrador Retrievers negative for MCD at the age of six to 17 weeks., Clinical Significance: These findings demonstrate that the development of MCD in the Labrador Retrievers in our study was not related to any disturbance in the development of the antebrachia and elbow joints during the rapid growth phase.

The aetiopathogenesis of medial coronoid disease (MCD) remains obscure, despite its high prevalence. The role of changes to subchondral bone or articular cartilage is much debated. Although there is evidence of micro-damage to subchondral bone, it is not known whether this is a cause or a consequence of MCD, nor is it known whether articular cartilage is modified in the early stages of the disease. The aim of the present study was to use equilibrium partitioning of an ionic contrast agent with micro-computed tomography (microCT) to investigate changes to both the articular cartilage and the subchondral bone of the medial coronoid processes (MCP) of growing Labrador retrievers at an early stage of the disease and at different bodyweights. Of 14 purpose-bred Labrador retrievers (15-27 weeks), six were diagnosed with bilateral MCD and one was diagnosed with unilateral MCD on the basis of microCT studies. The mean X-ray attenuation of articular cartilage was significantly higher in dogs with MCD than in dogs without MCD (P<0.01). In all dogs, the mean X-ray attenuation of articular cartilage was significantly higher at the lateral (P<0.001) than at the proximal aspect of the MCP, indicating decreased glycosaminoglycan content. Changes in parameters of subchondral bone micro-architecture, namely the ratio of bone volume to tissue volume (BV/TV), bone surface density (BS/TV), bone surface to volume ratio (BS/BV), trabecular thickness (Tb.Th; mm), size of marrow cavities described by trabecular spacing (Tb.Sp; mm), and structural model index (SMI), differed significantly by litter (P<0.05) due to the difference in age and weight, but not by the presence/absence of MCD (P>0.05), indicating that subchondral bone density is not affected in early MCD. This study demonstrated that cartilage matrix and not subchondral bone density is affected in the early stages of MCD., (Copyright © 2013 Elsevier Ltd. All rights reserved.)