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Abstract Frozen shoulder is a spontaneously self-resolving chronic inflammatory fibrotic human disease, which distinguishes the condition from most fibrotic diseases that are progressive and irreversible. Using single-cell analysis, we identify pro-inflammatory MERTKlowCD48+ macrophages and MERTK + LYVE1 + MRC1+ macrophages enriched for negative regulators of inflammation which co-exist in frozen shoulder capsule tissues. Micro-cultures of patient-derived cells identify integrin-mediated cell-matrix interactions between MERTK+ macrophages and pro-resolving DKK3+ and POSTN+ fibroblasts, suggesting that matrix remodelling plays a role in frozen shoulder resolution. Cross-tissue analysis reveals a shared gene expression cassette between shoulder capsule MERTK+ macrophages and a respective population enriched in synovial tissues of rheumatoid arthritis patients in disease remission, supporting the concept that MERTK+ macrophages mediate resolution of inflammation and fibrosis. Single-cell transcriptomic profiling and spatial analysis of human foetal shoulder tissues identify MERTK + LYVE1 + MRC1+ macrophages and DKK3+ and POSTN+ fibroblast populations analogous to those in frozen shoulder, suggesting that the template to resolve fibrosis is established during shoulder development. Crosstalk between MerTK+ macrophages and pro-resolving DKK3+ and POSTN+ fibroblasts could facilitate resolution of frozen shoulder, providing a basis for potential therapeutic resolution of persistent fibrotic diseases.

Blastocystis sp. is a gastrointestinal protozoan commonly encountered in humans and animals. Specificity to certain hosts may be associated with 38 known subtypes (STs) and 8 nonmammalian and avian STs (NMASTs). This can be determined by analyzing ST–host associations, ST–allele data, genetic variability analyses, and fixation index (FST) with sufficient data present. Thus, newly acquired and previously published data on Blastocystis sp. STs and NMASTs from the Philippines were compiled to determine the following: (1) ST–host associations, (2) ST–allele diversity per ST in certain hosts/sources, (3) intrasubtype diversity of certain STs found in different hosts using genetic variability analysis, and (4) comparison of similarities between specific ST populations to determine if these are the same circulating populations using FST. A total of 448 samples subtyped using both sequence-tagged site primers and the 600-bp barcoding region of the Blastocystis sp. SSU rRNA gene were analyzed in this study. Patterns of association for the Philippine samples were similar to those from neighboring Southeast Asian countries and around the world: ST1–ST4 were found in humans but ST3 was the most common, ST5 were found in pigs, and ST6 and ST7 were found in poultry. Blastocystis sp. from humans are mostly the same ST alleles (ST3 allele 34 and ST1 allele 4) while 3–5 ST alleles were found in the most common STs in pigs, macaques, and poultry. Also, ST1, ST3, ST5, and NMAST I are undergoing population expansion according to genetic variability analyses through possible addition of new alleles based on ST–allele diversity. Moreover, FST shows the same circulating population of ST1 in humans, pigs, and water indicating a possible waterborne route of cross-transmission. In contrast, ST3 found in humans possibly come from the same circulating population and is genetically distinct from those in nonhuman sources.

The extended-spectrum β-lactamase (ESBL)-producing Escherichia coli is becoming a global public health concern. More comprehensive surveillance of β-lactam resistance in E. coli would improve monitoring strategies and control resistance transmission in contaminated environments. This study investigated the prevalence of β-lactamase genes in E. coli isolated from the Seven Crater Lakes in San Pablo, Laguna, Philippines. Water samples from lakes were collected for the isolation of E. coli (n = 846) and molecular characterization by detecting the presence of the uidA gene. The isolates were then tested for the presence of β-lactamase genes using PCR. Among the screened genes, blaAmpC was the most dominant (91%). Other β-lactamase genes such as blaTEM, blaSHV, and blaCTXM were also detected with percentage occurrence of 34, 5, and 1%, respectively. Multiple genes within individual isolates were also observed, wherein blaTEM/AmpC was the most prevalent gene combination. Moreover, a significant negative correlation between blaAmpC with blaSHV and blaCTXM was depicted in this study. Overall, these findings demonstrate the presence of β-lactamase genes in E. coli in the Seven Crater Lakes of San Pablo and can be used in developing effective strategies to control antibiotic resistance in environmental waters. HIGHLIGHTS blaAmpC was the predominant gene detected among the five screened β-lactamase genes.; A significant association was detected between blaAmpC with blaSHV and blaCTXM.; blaKPC was not detected in any E. coli isolates from the lakes.; Seasonal variations have an effect on the fecal coliform counts and occurrence of β-lactamase-resistant E. coli isolates.;

Abstract Objectives Osteoarthritis (OA) is increasingly recognised as a whole joint disease, with an important role for synovium. However, the repertoire of immune cells and fibroblasts that constitute OA synovium remains understudied. This study aims to characterise the cellular composition of advanced OA synovium and to explore potential correlations between different cell types and patient demographics or clinical scores. Methods Synovium, collected from 10 patients with advanced OA during total knee replacement surgery, was collagenase-digested, and cells were stained for flow cytometry analysis. Formalin-fixed paraffin-embedded synovium was sectioned, stained with immunofluorescence, and imaged using the multiplex Cell DIVE platform. Patient demographics and clinical scores were also collected. Results The proportion of immune cells in OA synovium varied between patients (8–38% of all cells). Macrophages and T cells were the dominant immune cell populations, together representing 76% of immune cells. Age positively correlated with the proportion of macrophages, and negatively correlated with T cells. CCR6+ T cells were found in 6/10 patients; these patients had a higher mean Kellgren-Lawrence grade across the three knee compartments. Immunofluorescence staining showed that macrophages were present in the lining as well as distributed throughout the sublining, while T and B cells were mainly localised near vessels in the sublining. Fibroblast subsets (CD45−PDPN+) based on the expression of CD34/CD90 or FAP/CD90 were identified in all patient samples, and some populations correlate with the percentage of immune cells or clinical scores. Immunofluorescence staining showed that FAP expression was particularly strong in the lining layer, but also present throughout the sublining layer. CD90 expression was exclusively found around vessels in the sublining, while CD34 was mostly found in the sublining but also occasionally in the lining layer. Conclusions There are significant differences in the relative proportions and subsets of immune cells in OA synovium; exploratory correlative analyses suggest that these differences might be correlated with age, clinical scores, or fibroblast subsets. Additional studies are required to understand how different cell types affect OA pathobiology, and if the presence or proportion of cell subsets relates to disease phenotypes.

The integration of next-generation sequencing into the identification and characterization of resistant and virulent strains as well as the routine surveillance of foodborne pathogens such as Salmonella enterica have not yet been accomplished in the Philippines. This study investigated the antimicrobial profiles, virulence, and susceptibility of the 105 S. enterica isolates from swine and chicken samples obtained from slaughterhouses and public wet markets in Metropolitan Manila using whole-genome sequence analysis. Four predominant serovars were identified in genotypic serotyping, namely, Infantis (26.7%), Anatum (19.1%), Rissen (18.1%), and London (13.3%). Phenotypic antimicrobial resistance (AMR) profiling revealed that 65% of the isolates were resistant to at least one antibiotic, 37% were multidrug resistant (MDR), and 57% were extended-spectrum β-lactamase producers. Bioinformatic analysis revealed that isolates had resistance genes and plasmids belonging to the Col and Inc plasmid families that confer resistance against tetracycline (64%), sulfonamide (56%), and streptomycin (56%). Further analyses revealed the presence of 155 virulence genes, 42 of which were serovar-specific. The virulence genes primarily code for host immune system modulators, iron acquisition enzyme complexes, host cell invasion proteins, as well as proteins that allow intracellular and intramacrophage survival. This study showed that virulent MDR S. enterica and several phenotypic and genotypic AMR patterns were present in the food chain. It serves as a foundation to understand the current AMR status in the Philippines food chain and to prompt the creation of preventative measures and efficient treatments against foodborne pathogens.

Abstract Background Salmonella are pathogenic foodborne bacteria with complex pathogenicity from numerous virulence genes housed in Salmonella pathogenicity islands (SPIs), plasmids, and other gene cassettes. However, Salmonella virulence gene distributions and mechanisms remain unestablished. In the Philippines, studies mainly report Salmonella incidences and antimicrobial resistance, but little to none on virulence profiles, their associations to animal sources, collection sites and Salmonella serogroups. Hence, a total of 799 Salmonella isolates, previously obtained from pig, cow, and chicken meat samples in wet markets and abattoirs (wet markets: 124 chicken, 151 cow, and 352 pig meat isolates; abattoirs: 172 pig tonsil and jejunum isolates) in Metro Manila, Philippines, were revived and confirmed as Salmonella through invA gene polymerase chain reaction (PCR). Isolates were then screened for eight virulence genes, namely avrA, hilA, sseC, mgtC, spi4R, pipB, spvC and spvR, by optimized multiplex PCR and significant pair associations between virulence genes were determined through Fisher’s exact test. Gene frequency patterns were also determined. Salmonella serogroups in addition to animal sources and location types were also used to predict virulence genes prevalence using binary logistic regression. Results High frequencies (64 to 98%) of SPI virulence genes were detected among 799 Salmonella isolates namely mgtC, pipB, avrA, hilA, spi4R and sseC, from most to least. However, only one isolate was positive for plasmid-borne virulence genes, spvC and spvR. Diversity in virulence genes across Salmonella serogroups for 587 Salmonella isolates (O:3 = 250, O:4 = 133, O:6,7 = 99, O:8 = 93, O:9 = 12) was also demonstrated through statistical predictions, particularly for avrA, hilA, sseC, and mgtC. mgtC, the most frequent virulence gene, was predicted by serogroup O:9, while sseC, the least frequent, was predicted by serogroup O:4 and chicken animal source. The highest virulence gene pattern involved SPIs 1-5 genes which suggests the wide distribution and high pathogenic potential of Salmonella. Statistical analyses showed five virulence gene pair associations, namely avrA and hilA, avrA and spi4R, hilA and spi4R, sseC and spi4R, and mgtC and pipB. The animal sources predicted the presence of virulence genes, sseC and pipB, whereas location type for hilA and spi4R, suggesting that these factors may contribute to the type and pathogenicity of Salmonella present. Conclusion The high prevalence of virulence genes among Salmonella in the study suggests the high pathogenic potential of Salmonella from abattoirs and wet markets of Metro Manila, Philippines which poses food safety and public health concerns and threatens the Philippine food animal industry. Statistical associations between virulence genes and prediction analyses across Salmonella serogroups and external factors such as animal source and location type and presence of virulence genes suggest the diversity of Salmonella virulence and illustrate determining factors to Salmonella pathogenicity. This study recommends relevant agencies in the Philippines to improve standards in food animal industries and increase efforts in monitoring of foodborne pathogens.

Gold nanoparticles (AuNPs) are widely used in biomedicine and their specific properties including, size, geometrics, and surface coating, will affect their fate and behaviour in biological systems. These properties are well studied for their intended biological targets, but there is a lack of understanding on the mechanisms by which AuNPs interact in non-target organisms when they enter the environment. We investigated the effects of size and surface chemistry of AuNPs on their bioavailability, tissue distribution and potential toxicity using zebrafish (Danio rerio) as an experimental model. Larval zebrafish were exposed to fluorescently tagged AuNPs of different sizes (10–100 nm) and surface modifications (TNFα, NHS/PAMAM and PEG), and uptake, tissue distribution and depuration rates were measured using selective-plane illumination microscopy (SPIM). The gut and pronephric tubules were found to contain detectable levels of AuNPs, and the concentration-dependent accumulation was related to the particle size. Surface addition of PEG and TNFα appeared to enhance particle accumulation in the pronephric tubules compared to uncoated particles. Depuration studies showed a gradual removal of particles from the gut and pronephric tubules, although fluorescence indicating the presence of the AuNPs remained in the pronephros 96 h after exposure. Toxicity assessment using two transgenic zebrafish reporter lines, however, revealed no AuNP-related renal injury or cellular oxidative stress. Collectively, our data show that AuNPs used in medical applications across the size range 40–80 nm, are bioavailable to larval zebrafish and some may persist in renal tissue, although their presence did not result in measurable toxicity with respect to pronephric organ function or cellular oxidative stress for short term exposures.

This article investigates the challenges of the application of international law in a domestic setting as depicted in the ongoing trial of Thomas Kwoyelo. Kwoyelo, a former child soldier and commander in the Lord's Resistance Army, is being prosecuted for the commission inter alia of murder, kidnapping with intent to murder, and pillaging, all as war crimes and crimes against humanity under International Humanitarian Law, Customary International Law and the Penal Code Act of Uganda. He is currently standing trial at the International Crimes Division of the High Court of Uganda. This trial is a unique test for the Ugandan judiciary, as it is faced with its first prosecution of an individual charged with crimes under international law. In a bid to apply international law domestically the Court has faced several challenges which have disabled the progress of the trial on many levels, arguably at the expense of the accused and the victims at large. The article primarily seeks to analyse the capability of the court to prosecute Kwoyelo for the commission of international crimes as well as to examine the challenges faced by the Court. The history of the conflict will be examined. This will be followed by an overview of the judicial hurdles faced by the Ugandan State in eventually charging Kwoyelo. The article will then analyse the present challenges faced by the Court. Finally, several recommendations are offered.

Laguna Lake is an economically important resource in the Philippines, with reports of declining water quality due to fecal pollution. Currently, monitoring methods rely on counting fecal indicator bacteria, which does not supply information on potential sources of contamination. In this study, we predicted sources of Escherichia coli in lake stations and tributaries by establishing a fecal source library composed of rep-PCR DNA fingerprints of human, cattle, swine, poultry, and sewage samples (n = 1,408). We also evaluated three statistical methods for predicting fecal contamination sources in surface waters. Random forest (RF) outperformed k-nearest neighbors and discriminant analysis of principal components in terms of average rates of correct classification in two- (84.85%), three- (82.45%), and five-way (74.77%) categorical splits. Overall, RF exhibited the most balanced prediction, which is crucial for disproportionate libraries. Source tracking of environmental isolates (n = 332) revealed the dominance of sewage (47.59%) followed by human sources (29.22%), poultry (12.65%), swine (7.23%), and cattle (3.31%) using RF. This study demonstrates the promising utility of a library-dependent method in augmenting current monitoring systems for source attribution of fecal contamination in Laguna Lake. This is also the first known report of microbial source tracking using rep-PCR conducted in surface waters of the Laguna Lake watershed. HIGHLIGHTS DNA fingerprinting of E. coli, coupled with machine learning algorithms, can be used to discriminate fecal pollution sources in Laguna Lake, Philippines.; The majority of E. coli isolates can be attributed to sewage contamination, followed by human and agricultural sources.; Source-tracking methods can empower local agencies responsible for water quality management to minimize public health and economic risks.;

Laguna Lake is the largest inland freshwater body in the Philippines. Although it is classified to be usable for agricultural and recreational purposes by the country's Department of Environment and Natural Resources (DENR), studies looking at lake ecology revealed severe fecal contamination which contributes to the deterioration of water quality. Determining the sources of fecal contamination is necessary for lake protection and management. This study utilized a library-independent method of microbial source tracking (LIM-MST) to identify sources of fecal contamination in selected Laguna Lake stations and tributaries. Genetic markers of the host-associated Escherichia coli, heat-labile toxin (LTIIA) and heat-stable II (STII), were used to identify cattle and swine fecal contaminations, respectively. Meanwhile, human mitochondrial DNA (mtDNA) was used to identify human fecal contamination. Results identified the presence of agricultural and human fecal contamination in Laguna Lake Stations 1 and 5, Mangangate River, and Alabang River. The selected sites are known to be surrounded by residential and industrial complexes, and most of their discharges find their way into the lake. The identification of the specific sources of fecal contamination will guide management practices that aim to regulate the discharges in order to improve the water quality of Laguna Lake. HIGHLIGHTS The Escherichia coli LTIIa and STII genes, as well as human NADH mtDNA, are useful biomarkers for detecting fecal contamination in Laguna Lake.; Fecal contamination from agricultural and human origins can be detected in some lake stations and rivers near Laguna Lake.; As indicated by the high detection of LTIIa gene, fecal contamination in the sampling sites was mostly of cattle origin.;

Lakes are one of the sinks of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs); however, information on ARB and ARGs in lakes in the Philippines is scarce. In this study, Escherichia coli was isolated from the largest freshwater lake in the Philippines, Laguna Lake, to detect antibiotic resistance and the presence of ARGs. Broth microdilution assay (BMA) and molecular identification of five environmentally prevalent ARGs (strA, blaCTX-M, blaSHV, blaTEM, and tetA) were performed. The majority (75.70%) of the isolates harbored at least one of the targeted antibiotic genes. Multiplex PCR detected about 49.07% of the isolates had genes for extended-spectrum β-lactamases (ESBL), which were mostly represented by blaTEM (47.66%). The genes strA and tetA were observed in this study with detection frequencies of 29.91 and 45.33%, respectively. About 95.69% of thermotolerant E. coli isolates were non-susceptible to six different antibiotics using BMA. Nearly 37% of the isolates were found to be multidrug-resistant (MDR) with most isolates resistant to ampicillin (81.72%). Furthermore, the occurrence of ESBL genes was significantly correlated with tetA genes (P = 0.013). To date, this study is the first to report on the presence of MDR and thermotolerant E. coli in Laguna Lake, Philippines. HIGHLIGHTS First report of the ecological presence of thermotolerant and multidrug-resistant Escherichia coli (37%) in Laguna Lake.; Most (75.70%) isolates harbored at least one of the targeted antibiotic resistance genes (strA, blaTEM, blaCTX-M, blaSHV, and tetA).; First report of the occurrence of aminoglycoside resistant gene (strA) and tetracycline efflux gene (tetA) in waterborne E. coli isolated in the Philippines.;

Water quality deterioration in source waters poses increased health, environmental, and economic risks. Here, we genotyped Cryptosporidium spp. obtained from water samples of Laguna Lake, Philippines, and its tributaries for the purpose of source-tracking fecal contamination. A total of 104 surface water samples were collected over a 1-year period (March 2018 to April 2019). Detection of Cryptosporidium was carried out using genus-specific primers targeting a fragment of the small subunit (SSU) rRNA gene. The study revealed 8 (14%) tributary samples and 1 (2.77%) lake sample positive for contamination. The species were determined to be C. parvum (n = 4), C. muris (n = 2), C. hominis (n = 1), C. galli (n = 1), C. baileyi (n = 1), C. suis (n = 1), as well as rat genotype IV (n = 1). Two species were detected in duck (C. baileyi) and cattle (C. parvum) fecal samples. The data presented suggest that Cryptosporidium contamination is likely to come from sewage or human feces as well as various agricultural sources (i.e. cattle, swine, and poultry). This information reveals the importance of mitigating fecal pollution in the lake system and minimizing health risks due to exposure to zoonotic Cryptosporidium species. HIGHLIGHTS Zoonotic and pathogenic species of Cryptosporidium in a freshwater reservoir point to domestic and agricultural sources of fecal contamination.; Cryptosporidium can be a potential marker for microbial source tracking in the Laguna Lake watershed to augment current monitoring efforts.;

Multiple sulfatase deficiency (MSD) is a rare recessively inherited Mendelian disorder that manifests with developmental delay, neurodegeneration, skeletal deformities, facial dysmorphism, congenital growth retardation, and other clinical signs. The disorder is caused by mutations in the SUMF1 gene, which encodes the formylglycine-generating enzyme (FGE), and responsible for the activation of sulfatases. Mutations in SUMF1 result in reduced or absent FGE function with consequent compromised activities of its client sulfatases. This leads to an accumulation of enzyme substrates, such as glycosaminoglycans and sulfolipids, within lysosomes and subsequently impaired lysosome function and cellular pathology. Currently, there are no disease modifying therapeutic options for MSD patients, hence the need for more suitable animal models to investigate the disorder. Here, we describe the characterisation of a sumf1 null zebrafish model, which has negligible sulfatase activity. Our sumf1−/− zebrafish model successfully recapitulates the pathology of MSD such as cranial malformation, altered bone development, an enlarged population of microglia, and growth retardation during early development but lacks early lethality of mouse Sumf1−/− models. Notably, we provide evidence of recovery in MSD pathology during later developmental stages, resulting in homozygous mutants that are viable. Hence, our data suggest the possibility of a unique compensatory mechanism that allows the sumf1−/− null zebrafish to survive better than human MSD patients and mouse Sumf1−/− models.

Flu, a viral infection caused by the influenza virus, is still a global public health concern with potential to cause seasonal epidemics and pandemics. Vaccination is considered the most effective protective strategy against the infection. However, given the high plasticity of the virus and the suboptimal immunogenicity of existing influenza vaccines, scientists are moving toward the development of universal vaccines. An important property of universal vaccines is their ability to induce heterosubtypic immunity, i.e., a wide immune response coverage toward different influenza subtypes. With the increasing number of studies and mounting evidence on the safety and efficacy of recombinant influenza vaccines (RIVs), they have been proposed as promising platforms for the development of universal vaccines. This review highlights the current progress and advances in the development of RIVs in the context of heterosubtypic immunity induction toward universal vaccine production. In particular, this review discussed existing knowledge on influenza and vaccine development, current hemagglutinin-based RIVs in the market and in the pipeline, other potential vaccine targets for RIVs (neuraminidase, matrix 1 and 2, nucleoprotein, polymerase acidic, and basic 1 and 2 antigens), and deantigenization process. This review also provided discussion points and future perspectives in looking at RIVs as potential universal vaccine candidates for influenza.

Sources of fecal contamination in Laguna Lake, Philippines, were identified using a library-independent microbial source tracking method targeting host-associated Bacteroides 16S rDNA-based markers. Water samples from nine lake stations were assessed for the presence of the fecal markers HF183 (human), BoBac (cattle), Pig-2-Bac (swine), and DuckBac (duck) from August 2019 to January 2020. HF183 (average concentration = 1.91 log10 copies/mL) was the most frequently detected, while Pig-2-Bac (average concentration = 2.47 log10 copies/mL) was the most abundant. The detected marker concentrations in different stations corresponded to the land use patterns around the lake. Generally, all marker concentrations were higher during the wet season (August–October), suggesting the effect of rainfall-associated factors on the movement and retention of markers from sources. There was a significant association (ρ = 0.45; p < 0.001) between phosphate and the concentration of HF183, suggesting domestic sewage-derived pollution. The markers had acceptable sensitivity and specificity, i.e., HF183 (S = 0.88; R = 0.99), Pig-2-Bac (S = 1.00; R = 1.00), and DuckBac (S = 0.94; R = 1.00), and therefore may be used for the continuous monitoring of fecal pollution in the lake and in designing interventions to improve the quality of the lake water.

Aptamers are nucleic acids that can bind with high affinity and specificity to a range of target molecules. However, their functionality relies on their secondary and tertiary structures such that the combination of nucleotides determines their three-dimensional conformation. In this study, the binding mechanisms of candidate aptamers and their interactions with selected target proteins found in the cell surface of Leptospira were predicted to select high-affinity aptamers. Four aptamers were evaluated through molecular modeling and docking using available software and web-based tools, following the workflow previously designed for in silico evaluation of DNA aptamers. The most predominant and highly conserved surface-exposed proteins among pathogenic Leptospira species were used as aptamer targets. The highest number of interactions was seen in aptamers AP5 and AP1. Hydrogen bonds, along with a few hydrophobic interactions, occur in most aptamer–protein complexes. Further analysis revealed serine, threonine, glutamine, and lysine as main protein residues. H-bond interactions occur mostly with polar amino acids, as reflected in the predicted interaction profiles of aptamer–protein complexes. In silico strategies allowed the identification of key residues crucial in aptamer–target interaction during aptamer screening. Such information can be used in aptamer modification for improved binding affinity and accuracy for diagnostics application.

The current gold standard for Salmonella serotyping is costly, labor-intensive and time-consuming. However, proper identification is key to monitor Salmonella transmission and implementation of necessary control measures. The onset of advanced molecular techniques has lessened resource and labor requirements; however, it still remains complex, unestablished and plagued with insufficiencies. Hence, a simpler serotyping method with sufficient resolution is needed. In this study, the invA virulence gene, associated with Salmonella invasion into host cells and is considered as a marker for Salmonella detection, was amplified and sequenced among isolates from meat samples in Metro Manila, Philippines. This was followed by sequence alignments with reference sequences (Refseqs), oversaturation and model tests, phylogenetic tree analyses and signal detections. Unfortunately, alignment of a 229bp amplified and sequenced invA gene segment with Refseqs generated little to no base variations and consequently provided insufficient phylogenetic resolution for molecular serotyping (0 of the 17 serotypes tested). However, another segment of 1498bp, outside the amplified region, showed considerable base variation in alignment and consequently resolved a maximum of 13 out of 17 (76.47%) serotypes tested, all generated trees considered. These suggest the potential of the invA virulence gene as a single-gene marker for molecular serotyping of Salmonella through phylogenetic analyses.

A robust monitoring network that provides quantitative information about the status of imperiled species at key life stages and geographic locations over time is fundamental for sustainable management of fisheries resources. For anadromous species, management actions in one geographic domain can substantially affect abundance of subsequent life stages that span broad geographic regions. Quantitative metrics (e.g., abundance, movement, survival, life history diversity, and condition) at multiple life stages are needed to inform how management actions (e.g., hatcheries, harvest, hydrology, and habitat restoration) influence salmon population dynamics. The existing monitoring network for endangered Sacramento River winter-run Chinook Salmon (SRWRC, Oncorhynchus tshawytscha) in California’s Central Valley was compared to conceptual models developed for each life stage and geographic region of the life cycle to identify relevant SRWRC metrics. We concluded that the current monitoring network was insufficient to diagnose when (life stage) and where (geographic domain) chronic or episodic reductions in SRWRC cohorts occur, precluding within- and among-year comparisons. The strongest quantitative data exist in the Upper Sacramento River, where abundance estimates are generated for adult spawners and emigrating juveniles. However, once SRWRC leave the upper river, our knowledge of their identity, abundance, and condition diminishes, despite the juvenile monitoring enterprise. We identified six system-wide recommended actions to strengthen the value of data generated from the existing monitoring network to assess resource management actions: (1) incorporate genetic run identification; (2) develop juvenile abundance estimates; (3) collect data for life history diversity metrics at multiple life stages; (4) expand and enhance real-time fish survival and movement monitoring; (5) collect fish condition data; and (6) provide timely public access to monitoring data in open data formats. To illustrate how updated technologies can enhance the existing monitoring to provide quantitative data on SRWRC, we provide examples of how each recommendation can address specific management issues

Genetic studies must enroll large numbers of participants to obtain adequate statistical power. Data are needed on how researchers can best use limited financial and practical resources to achieve these targets, especially in under-represented populations. This paper provides a retrospective analysis of the recruitment strategies for a large glaucoma genetics study in African Americans. The Primary Open-Angle African American Glaucoma Genetics study enrolled 10,192 African American subjects from the Philadelphia region. Major recruitment approaches included clinic enrollment from University of Pennsylvania (UPenn) sites, clinic enrollment from external sites, sampling of Penn Medicine Biobank (PMBB), and community outreach. We calculated the enrollment yield, cost per subject, and seasonal trends of these approaches. The majority (65%) of subject were enrolled from UPenn sites with an average cost of $133/subject. Over time, monthly case enrollment declined as the pool of eligible subjects was depleted. Expanding to external sites boosted case numbers ($129/subject) and the biobank provided additional controls at low cost ($5/subject), in large part due to the generosity of PMBB providing samples free of cost. Community outreach was costly with low return on enrollment ($978/subject for 220 subjects). Summer months (Jun-Aug) produced the highest recruitment yields (p

Abstract Background The stranding events of cetaceans in the Philippines provide opportunities for gathering biological information and specimens, especially from the pelagic forms. As part of an effort to monitor the health of wild cetaceans, this study detected Leptospira spp. and Toxoplasma gondii, causative agents of the emerging zoonotic diseases leptospirosis and toxoplasmosis respectively, in their stranded representatives. From October 2016–August 2018, 40 cetaceans (representing 14 species) that stranded nationwide were sampled for brain, cardiac muscle, skeletal muscle, kidney, and blood tissues, urine, and sera. These were subjected to molecular, serological, culture, and histopathological analyses to detect the target pathogens. Results T. gondii was detected in 20 (71%) of the 28 cetaceans with biological samples subjected to either molecular detection through RE gene amplification or IgG antibodies detection through agglutination-based serological assay. On the other hand, Leptospira was detected in 18 (64%) of 28 cetaceans with biological samples subjected to bacterial culture, molecular detection through 16S rDNA amplification, or IgM antibodies detection through ELISA-based serological assay. Conclusions There is the plausibility of toxoplasmosis and leptospirosis in cetacean populations found in the Philippines, however, acute or chronic phases of infections in sampled stranded individuals cannot be confirmed in the absence of supporting pathological observations and corroborating detection tests. Further studies should look for more evidences of pathogenicity, and explore the specific mechanisms by which pelagic cetacean species become infected by Leptospira spp. and T. gondii. As there is growing evidence on the role of cetaceans as sentinels of land-sea movement of emerging pathogens and the diseases they cause, any opportunity, such as their stranding events, should be maximized to investigate the health of their populations. Moreover, the role of leptospirosis or toxoplasmosis in these stranding events must be considered.

Ensuring efficient and clean combustion performance of liquid-fueled engines requires comprehension of the influence of fuel composition and properties on flame behavior, such as flame liftoff height (LOH) and lean blowout limit (LBO). Spray flame stability is strongly affected by both the fuel reactivity and physical properties. Herein, the flame stability mechanism represented by LOH is investigated for seven jet turbine fuels, including surrogate, alternative, and conventional jet fuels, using a laboratory spray burner. Based on the experimental observations, the current work introduces a new analysis, which provides insight into the competing/complementing processes that occur in a multi-phase reacting system and highlights the key properties important in spray flame dynamics, accounting for both the fuel spray/vaporization as well as the chemical reactivity, to explain the relative differences in LOH of complex multicomponent fuels. Results show that spray flame stabilization occurs when there is a balance between the local spray burning velocity and the incoming jet velocity, which is strongly associated with laminar flame speed and the relative amount of liquid and gaseous fuel crossing the flame preheat region. Using a multicomponent droplet evaporation model, it was observed that preferential vaporization of the lighter and more reactive species of the simple surrogate fuels contribute to a shorter lifted flame as compared to fuels consisting of heavier and/or less reactive components. The LOH of real jet fuels showed strong sensitivity to droplet vaporization and mixing, which is controlled by the fuels’ volatility (i.e., boiling temperature at 50% distillation volume) and atomized droplet size, and to a lesser extent the reactivity represented by laminar flame speed. The enhanced linearity in the correlation of 50% vapor distilled compared to the other distillation cuts suggests that preferential vaporization could play an important role in defining the LOH stabilization mechanism even for more complex fuels.

Due to the misuse and overuse of antibiotics, antibiotic residues accumulate in natural environments, leading to the development of antibiotic-resistant bacteria (ARBs). The presence of ARBs in bodies of water poses health hazards to the surrounding community. This study focused on Laguna Lake, the largest lake in the Philippines, which serves as a water source for agriculture and domestic purposes. We aimed to detect the presence of antibiotic-resistant Escherichia coli from the lake waters and potential reservoirs of resistance as well as determine the multiple antibiotic resistance (MAR) indices of the isolates. E. coli (n = 450) was isolated from fecal-associated samples (chicken, cow, pig, human, sewage) and water samples (sites in Laguna Lake and selected river tributaries). The isolates were subjected to an antibiotic resistance assay using VITEK 2®. Among the 16 antibiotics tested, the isolates exhibited varying resistance to 14, but complete susceptibility to amikacin and tigecycline was observed. Isolates were most frequently resistant to ampicillin (196/450, 43.6%). Among fecal-associated samples, chicken isolates exhibited the highest MAR index (0.174), whereas samples from Pila River exhibited the highest MAR index (0.152) among water samples. The results of this study demonstrate the presence of multidrug-resistant E. coli in samples collected around Laguna Lake and reveal fecal and sewage sources as potential reservoirs of ARBs in the water body. With this information, the public is urged to use antibiotics responsibly to help mitigate the spread of antibiotic resistance.

Rapid and safe access to new plant genetic stocks is crucial for primary plant industries to remain profitable, sustainable, and internationally competitive. Imported plant species may spend several years in Post Entry Quarantine (PEQ) facilities, undergoing pathogen testing which can impact the ability of plant industries to quickly adapt to new global market opportunities by accessing new varieties. Advances in high throughput sequencing (HTS) technologies provide new opportunities for a broad range of fields, including phytosanitary diagnostics. In this study, we compare the performance of two HTS methods (RNA-Seq and sRNA-Seq) with that of existing PEQ molecular assays in detecting and identifying viruses and viroids from various plant commodities. To analyze the data, we tested several bioinformatics tools which rely on different approaches, including direct-read, de novo, and reference-guided assembly. We implemented VirusReport, a new portable, scalable, and reproducible nextflow pipeline that analyses sRNA datasets to detect and identify viruses and viroids. We raise awareness of the need to evaluate cross-sample contamination when analyzing HTS data routinely and of using methods to mitigate index cross-talk. Overall, our results suggest that sRNA analyzed using VirReport provides opportunities to improve quarantine testing at PEQ by detecting all regulated exotic viruses from imported plants in a single assay.

Since early legal, medical and scientific discussions of homosexuality, the discipline of psychology has assumed an instrumental role in both maintaining and challenging cultural and political perspectives on same-sex desire, identity and behaviour. This article presents a critical historical review of psychological research on same-sex desire in the United States, with a focus on the way in which studies reflect three master narratives on the nature and meaning of same-sex attraction: (1) a sickness script that dominated the majority of research from the late 1800s to the 1970s; (2) a species script that became popular with the removal of homosexuality as a diagnosable mental illness in 1973 and initiated several new lines of research; and (3) a subject script inspired by scholarly and cultural shifts beginning in the 1990s that challenged the taxonomy of sexual identity. We conclude with a discussion of the transformative potential of a narrative approach that integrates analysis of historical forces and individual psychological development. © 2013 Copyright Taylor and Francis Group, LLC.

The constitutional right to privacy is enshrined in section 14 of the Constitution of the Republic of South Africa, 1996. It is premised on the notion that all persons should be protected from intrusions on their privacy by any person or institution. The Constitutional Court has also, on numerous occasions, held that the right to privacy is bolstered by its connection with the right to human dignity. It is undeniable that every person's right to privacy should be protected. However, a person's right to privacy is violated when police officials conduct warrantless search and seizure operations. Generally section 22 of the Criminal Procedure Act provides for warrantless search and seizure operations when a police official has a reasonable suspicion that a search warrant will be issued to him and that a delay in obtaining such a warrant would defeat the object of the search. Warrantless searches are important for the prevention of crime, but recent case law has suggested that there has been a progressive shift towards protecting the right to privacy of the individual subjected to warrantless searches, since there are a number of laws besides section 22 that regulate warrantless searches and which have been declared to be constitutionally invalid. This article seeks to demonstrate that the current regulatory framework for warrantless searches should be reviewed in order to protect the legitimacy of the police as well as the dignity and privacy of the citizens of South Africa.

Despite the major threat of leptospirosis to public health in the Philippines, its epidemiologic data remain scarce. Multilocus sequence typing (MLST) is a method often used for identification of circulating Leptospira species and disease surveillance. Unfortunately, molecular typing of Leptospira isolates is not routinely done in most hospital settings. A simplified MLST scheme targeting three loci (adk, lipL41, mreA) was performed for rapid direct typing of Leptospira in clinical specimens. Blood samples from suspected or clinically diagnosed cases (n = 50) were initially screened via polymerase chain reaction (PCR) targeting 23S rRNA, 16S rRNA (rrs2), and lipL32 genes. From the nine positives, seven had interpretable data from MLST. Allelic profiles identified L. interrogans in all positive samples. Six were assigned to ST12 of serovar Manilae (serogroup Pyrogenes) while one sample cannot be clearly differentiated between two serovars/serogroups, Bataviae/Losbanos (serogroup Bataviae) or Australis (serogroup Australis), indicating possibility of a new ST. Phylogenetic analysis confirmed that the application of simplified MLST scheme produces consistent results with the seven-loci genetic profile of published Leptospira MLST schemes. Reduced scheme addressed the challenges often encountered in the amplification of full MLST genetic profile of Leptospira. The approach is a potential alternative to serological tests for rapid typing of clinical specimens and can also aid in investigations on disease epidemiology specifically to monitor occurrence, pathogen transmission, host specificity and susceptibility, and other factors that could lead to potential outbreaks.

The relatively high frequency of marine mammal stranding events in the Philippines provide many research opportunities. A select set of stranders (n = 21) from 2017 to 2018 were sampled for bacteriology and histopathology. Pertinent tissues and bacteria were collected from individuals representing eight cetacean species (i.e. Feresa attenuata, Kogia breviceps, Globicephala macrorhynchus, Grampus griseus, Lagenodelphis hosei, Peponocephala electra, Stenella attenuata and Stenella longirostris) and were subjected to histopathological examination and antibiotic resistance screening, respectively. The antibiotic resistance profiles of 24 bacteria (belonging to genera Escherichia, Enterobacter, Klebsiella, Proteus, and Shigella) that were isolated from four cetaceans were determined using 18 antibiotics. All 24 isolates were resistant to at least one antibiotic class, and 79.17% were classified as multiple antibiotic resistant (MAR). The MAR index values of isolates ranged from 0.06 to 0.39 with all the isolates resistant to erythromycin (100%; n = 24) and susceptible to imipenem, doripenem, ciprofloxacin, chloramphenicol, and gentamicin (100%; n = 24). The resistance profiles of these bacteria show the extent of antimicrobial resistance in the marine environment, and may inform medical management decisions during rehabilitation of stranded cetaceans. Due to inadequate gross descriptions and limited data gathered by the responders during the stranding events, the significance of histopathological lesions in association with disease diagnosis in each cetacean stranding or mortality remained inconclusive; however, these histopathological findings may be indicative or contributory to the resulting debility and stress during their strandings. The findings of the study demonstrate the challenges faced by cetacean species in the wild, such as but not limited to, biological pollution through land-sea movement of effluents, fisheries interactions, and anthropogenic activities.

Salmonella enterica is known as one of the most common foodborne pathogens worldwide. While salmonellosis is usually self-limiting, severe infections may require antimicrobial therapy. However, increasing resistance of Salmonella to antimicrobials, particularly fluoroquinolones and cephalosporins, is of utmost concern. The present study aimed to investigate the antimicrobial susceptibility of S. enterica isolated from pork, the major product in Philippine livestock production. Our results show that both the qnrS and the blaTEM antimicrobial resistance genes were present in 61.2% of the isolates. While qnrA (12.9%) and qnrB (39.3%) were found less frequently, co-carriage of blaTEM and one to three qnr subtypes was observed in 45.5% of the isolates. Co-carriage of blaTEM and blaCTX-M was also observed in 3.9% of the isolates. Antimicrobial susceptibility testing revealed that the majority of isolates were non-susceptible to ampicillin and trimethoprim/sulfamethoxazole, and 13.5% of the isolates were multidrug-resistant (MDR). MDR isolates belonged to either O:3,10, O:4, or an unidentified serogroup. High numbers of S. enterica carrying antimicrobial resistance genes (ARG), specifically the presence of isolates co-carrying resistance to both β-lactams and fluoroquinolones, raise a concern on antimicrobial use in the Philippine hog industry and on possible transmission of ARG to other bacteria.

This study aimed to detect Salmonella from retail meat collected from nine wet markets in Metro Manila, and identify the subtypes of Salmonella isolates using molecular serotyping assays from previously developed primers. Of the 720 collected meat samples, 57.64% were found to be Salmonella-contaminated. The most predominant serogroup was Salmonella O:3, and Salmonella serogroups O:4, O:6,7, O:8, O:9, and undetermined serogroups were also found. Most frequently detected isolates in bovine meat were S. 3:e,h:1,6 (putative identity: S. Anatum) and S: 4:e,h:1,2 (putative identity: S. Saintpaul), in porcine meat was S. 3:e,h:1,6 (putative identity: S. Anatum), and S. 8:i:z6 (putative identity: S. Kentucky) was common in poultry products. This study also demonstrated retail meat samples were contaminated with multiple Salmonella serogroups and serovars. This is the first Philippine study that utilized PCR-based assays to characterize Salmonella isolates down to a serovar level and provides baseline information regarding Salmonella prevalence and serovar distribution in retail meat. Molecular serotyping performed in this study can be used as an alternative approach to traditional serotyping in surveillance of Salmonella in the Philippines since the latter is expensive, time-consuming, and requires skilled technicians.

The violent nature of some crimes and the high crime rate in South Africa reflect the fact that some offenders constitute a real threat to the security of communities. It is understandable, therefore, that the state seeks to protect its citizens through preventive measures. Although South Africa has certain legal provisions on its statutory books, it seems that the declaration of persons as dangerous criminals is under-utilised. South African legislation dealing with the declaration of dangerous criminals can be improved by borrowing some traits of the Canadian legislation. Such features include the restriction of courts' discretion and the provision of concrete and more detailed guidelines on the nature of the offences for which the provision can be applied. The courts could also take into account the type of criminal history of the offender which would merit the declaration of a dangerous criminal. It is also important that the extent of the violence in an offence should be thoroughly defined in court. Courts need to balance their wide discretion on the matter with the provisions in the Act in order to protect the community against dangerous criminals.

Background/Purpose: The flagellated protozoon Trichomonas vaginalis that parasitizes the urogenital tract of humans was reported to harbor double-stranded RNA (dsRNA) viruses. These viruses, identified as Trichomonas vaginalis virus (TVV), belong to the genus Trichomonasvirus of the family Totiviridae. Four species, formally recognized by the International Committee on Taxonomy of Viruses (ICTV), have been reported and distinguished by pairwise comparisons of the sequences of genes coding for major capsid protein (CP) and RNA-dependent RNA polymerase (RdRp). Methods: Reverse transcription polymerase chain reaction (RT-PCR) was used to amplify the complimentary DNA of target virus genes coding for CP and RdRp. Sequence analyses confirmed the identity of the TVV isolates from T. vaginalis cultures. Results: A total of 35 dsRNA viruses were identified from 18 (19%) T. vaginalis isolates. Multiple TVV species were observed in six of the 18 T. vaginalis cultures. Phylogenetic analyses show monophyly in TVV1 and TVV2 whereas TVV3 and TVV4 appear paraphyletic. The phylogeny of Philippine Trichomonasvirus reflects the global distribution of its host. Conclusion: This is the first study in the Philippines and one of the two reports worldwide to detect the four TVVs and their concurrent infection in a single T. vaginalis isolate.

Abstract Background African Americans have been historically under-represented in genetic studies. More research is needed on effective recruitment strategies for this population, especially on approaches that supplement traditional clinic enrollment. This study evaluates the cost and efficacy of four supplemental recruitment methods employed by the Primary Open-Angle African American Glaucoma Genetics (POAAGG) study. Methods After enrolling 2304 patients from University of Pennsylvania ophthalmology clinics, the POAAGG study implemented four new recruitment methods to supplement clinic enrollment. These methods included: 1) outreach in the local community, 2) in-house screening of community members (“in-reach”), 3) expansion to two external sites, and 4) sampling of the Penn Medicine Biobank. The cost per subject was calculated for each method and enrollment among cases, controls, and suspects was reported. Results The biobank offered the lowest cost ($5/subject) and highest enrollment yield (n = 2073) of the four methods, but provided very few glaucoma cases (n = 31). External sites provided 88% of cases recruited from the four methods (n = 388; $85/subject), but case enrollment at these sites declined over the next 9 months as the pool of eligible subjects was depleted. Outreach and in-reach screenings of community members were very high cost for low return on enrollment ($569/subject for 102 subjects and $606/subject for 45 subjects, respectively). Conclusions The biobank offered the most cost-effective method for control enrollment, while expansion to external sites was necessary to recruit richly phenotyped cases. These recruitment methods helped the POAAGG study to exceed enrollment of the discovery cohort (n = 5500) 6 months in advance of the predicated deadline and could be adopted by other large genetic studies seeking to supplement clinic enrollment.

This data article contains transmission electron microscopy (TEM) images of psychrotolerant bacterium Shewanella olleyana sp. nov. Cells of S. olleyana were grown following an optimized culture conditions in liquid medium. Procedure for the preparation of cells suitable for TEM is described in detail.

The purpose of this study was to investigate the association between gender and primary open-angle glaucoma (POAG) among African Americans and to assess demographic, systemic, and behavioral factors that may contribute to differences between genders. The Primary Open-Angle African American Glaucoma Genetics (POAAGG) study had a case-control design and included African Americans 35 years and older, recruited from the greater Philadelphia, Pennsylvania. Diagnosis of POAG was based on evidence of both glaucomatous optic nerve damage and characteristic visual field loss. Demographic and behavioral information, history of systemic diseases and anthropometric measurements were obtained at study enrollment. Gender differences in risk of POAG were examined using multivariate logistic regression. A total of 2,290 POAG cases and 2,538 controls were included in the study. The percentage of men among cases was higher than among controls (38.6% vs 30.3%, P

doi: https://doi.org/10.15447/sfews.2017v15iss3art1A robust monitoring network that provides quantitative information about the status of imperiled species at key life stages and geographic locations over time is fundamental for sustainable management of fisheries resources. For anadromous species, management actions in one geographic domain can substantially affect abundance of subsequent life stages that span broad geographic regions. Quantitative metrics (e.g., abundance, movement, survival, life history diversity, and condition) at multiple life stages are needed to inform how management actions (e.g., hatcheries, harvest, hydrology, and habitat restoration) influence salmon population dynamics. The existing monitoring network for endangered Sacramento River winter-run Chinook Salmon (SRWRC, Oncorhynchus tshawytscha) in California’s Central Valley was compared to conceptual models developed for each life stage and geographic region of the life cycle to identify relevant SRWRC metrics. We concluded that the current monitoring network was insufficient to diagnose when (life stage) and where (geographic domain) chronic or episodic reductions in SRWRC cohorts occur, precluding within- and among-year comparisons. The strongest quantitative data exist in the Upper Sacramento River, where abundance estimates are generated for adult spawners and emigrating juveniles. However, once SRWRC leave the upper river, our knowledge of their identity, abundance, and condition diminishes, despite the juvenile monitoring enterprise. We identified six system-wide recommended actions to strengthen the value of data generated from the existing monitoring network to assess resource management actions: (1) incorporate genetic run identification; (2) develop juvenile abundance estimates; (3) collect data for life history diversity metrics at multiple life stages; (4) expand and enhance real-time fish survival and movement monitoring; (5) collect fish condition data; and (6) provide timely public access to monitoring data in open data formats. To illustrate how updated technologies can enhance the existing monitoring to provide quantitative data on SRWRC, we provide examples of how each recommendation can address specific management issues

Objective: To investigate the antibacterial activities of crude ethanol extracts of 12 Philippine medicinal plants. Methods: Crude ethanol extracts from 12 Philippine medicinal plants were evaluated for their antibacterial activity against methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, extended spectrum β-lactamase-producing, carbapenem-resistant Enterobacteriaceae and metallo-β-lactamase-producing Pseudomonas aeruginosa and Acinetobacter baumannii. Results: The leaf extracts of Psidium guajava, Phyllanthus niruri, Ehretia microphylla and Piper betle (P. betle) showed antibacterial activity against the Gram-positive methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus. P. betle showed the highest antibacterial activity for these bacteria in the disk diffusion (16–33 mm inhibition diameter), minimum inhibitory concentration (19–156 μg/mL) and minimum bactericidal concentration (312 μg/mL) assays. P. betle leaf extracts only showed remarkable antibacterial activity for all the Gram-negative multidrug-resistant bacteria (extended spectrum β-lactamase-producing, carbapenem-resistant Enterobacteriaceae and metallo-β-lactamase-producing) in the disk diffusion (17–21 mm inhibition diameter), minimum inhibitory concentration (312–625 μg/mL) and minimum bactericidal concentration (312–625 μg/mL) assays. Conclusions: P. betle had the greatest potential value against both Gram-negative and Gram-positive multidrug-resistant bacteria. Favorable antagonistic activities were also exhibited by the ethanol extracts of Psidium guajava, Phyllanthus niruri and Ehretia microphylla.