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During the production of salmonids in aquaculture, it is common to observe growth-stunted individuals. The cause for the so-called “loser fish syndrome” is unclear, which needs further investigation. Here, we present and compare computer vision systems for the automatic detection and classification of loser fish in Atlantic salmon images taken in sea cages. We evaluated two end-to-end approaches (combined detection and classification) based on YoloV5 and YoloV7, and a two-stage approach based on transfer learning for detection and an ensemble of classifiers (e.g., linear perception, Adaline, C-support vector, K-nearest neighbours, and multi-layer perceptron) for classification. To our knowledge, the use of an ensemble of classifiers, considering consolidated classifiers proposed in the literature, has not been applied to this problem before. Classification entailed the assigning of every fish to a healthy and a loser class. The results of the automatic classification were compared to the reliability of human classification. The best-performing computer vision approach was based on YoloV7, which reached a precision score of 86.30%, a recall score of 71.75%, and an F1 score of 78.35%. YoloV5 presented a precision of 79.7%, while the two-stage approach reached a precision of 66.05%. Human classification had a substantial agreement strength (Fleiss’ Kappa score of 0.68), highlighting that evaluation by a human is subjective. Our proposed automatic detection and classification system will enable farmers and researchers to follow the abundance of losers throughout the production period. We provide our dataset of annotated salmon images for further research.

It is estimated that biofilms cause up to 80 per cent of all wound infections, necessitating both the use of antiseptics and the drainage of purulent discharge. In the inflamed and infected wound, it is sometimes difficult to recognise the presence of bacterial biofilms. Here we describe an example of a non-healing, inflamed superficial to partial thickness burn wound that appeared to be associated with biofilm. Special reference to the relationship among biofilm, inflammation and non-healing of burn wounds is illustrated in the case study given. This case study also explores the use of hypochlorous acid (HOCl) in an infected burn wound. [ABSTRACT FROM AUTHOR]

Key Clinical Message Light chain proximal tubulopathy (LCPT) is a rare type of monoclonal gammopathy of renal significance. Clinicians should consider LCPT in the differential diagnosis of patients with renal or proximal tubular dysfunction with monoclonal gammopathy. They should confirm diagnosis by renal biopsy and initiate chemotherapy before disease progression.

Wound healing by secondary intention is often difficult and costly to treat, with most patients being managed by private nurse practitioners (PNPs) or primary care wound clinics. Daily or less frequent dressings are the most common treatment method used where primary closure was not possible or following wound dehiscence after surgery. In such instances, wounds remain open for an extended time, thus making them prone to infection, biofilm formation and inflammation, which delays healing. Herein, we present case studies exploring the use of a pharmaceutical-grade hypochlorous acid (HOCl) solution to facilitate wound healing through secondary intention with limited scar formation. [ABSTRACT FROM AUTHOR]

The morphology of larvae is a key factor influencing their behaviour, performance and ultimately their survival. There is evidence indicating a significant morphological variability among broods, and that this may be related to the size or conditions of the mother. However, this maternal influence is not consistent across decapod crustaceans. Using 35 broods from different mothers of the crab Hemigrapsus takanoi collected in the same locality of inner Tokyo Bay and at the same time, we tested the hypothesis that there is a positive relationship between the size of the mother and the progeny’s morphology. Our results indicate that different patterns in the length of the lateral, rostral and dorsal spines differentiated two distinct morphogroups of larvae. These morphogroups were linked to the size of the mother, showing that larger mothers produced bigger larvae with longer carapace spines. It is possible that larger size and longer spines can influence swimming performance and predator avoidance, respectively. These relationships should be tested in future experimental studies.

Radiation therapy represents an important cornerstone in the treatment of numerous cancers, as evidenced by the fact that approximately 50% of patients with cancer will receive this form of treatment. In 95% of these individuals, radiation therapy causes some form of skin injury as high doses of radiation causes significant harm to healthy tissue and skin. Management of injuries due to ionising radiation is challenging, with injuries ranging from acute redness to full ulceration of the skin. This can cause considerable anxiety and discomfort to patients. This case study explores the use of hypochlorous acid (HOCl) in the treatment of acute radiation injury of the lower eyelid skin. [ABSTRACT FROM AUTHOR]

Background: Height is an important parameter of human in anthropology and forensic medicine. We can measure the height of an individual from forearm length (FAL). In this study, we found the relationship between FAL and height in medical students. Aims and Objective: The objective of the study was to find co-relation between FAL and height of the individuals (male and female medical students) and to obtain regression formula to determine the height from FAL. Materials and Methods: Height and FALs were measured in 200 medical students of 17–22 years age (100 males and 100 females) of N.R.S Medical College, Kolkata. For finding, the relationship between height and FAL linear regression analysis was used. Results: In our study, average height of the female students was 158.64±5.28 cm, whereas in male students average height was 168.97±6.33 cm. Mean FAL has been 26.63±1.72 and 23.72±1.44 cm, respectively, in case of male and female students. Difference of the mean height and mean length of forearm values between female and male students was statistically significant. The correlation coefficient (r) between height and FAL of dominant side has been, respectively, +0.93, +0.96, and +0.97 in case of male, female, and total students. According to the linear regression, there was a relation between height and FAL. Conclusion: From our study, it is evident that we may estimate the height of a person from measurement of FAL. In future studies may be carried out to estimate height of an individual from other body parameters also.

Background: The surface of the developing human brain undergoes morphological changes with the formation of different sulci during the fetal period. During the first and second-trimester fetal brain surface remain smooth, and between the 28th and 30th weeks of gestation, new gyri and sulci appear due to rapid growth of the fetal brain. Aims and Objectives: To study the time of appearance of sulci in different surfaces of the fetal brain, which will help estimate the gestational age of the fetus from the specimen of the fetal brain. Materials and Methods: About 108 cerebral hemispheres from 54 fetal brains were examined in the Department of Anatomy, Medical College, Kolkata, West Bengal, in 1 year from December 2016 to December 2017. Among 54 fetal brains, 21 were museum specimens of the Department of Anatomy and Forensic Medicine of Medical College, Kolkata, and the rest were fetal cadavers. For brains from fetal cadavers, after brain removal, it was fixed in 10% formalin. Fetal brains were divided into two groups – a) brains from fetuses with bodyweight up to 1000 g with a weight gap of 100 g among the groups. b) brains from fetuses with body weight >1001 g with a weight gap of 250 g among the groups. Sulci in the superolateral, medial, and inferior surfaces of the fetal brain were noted carefully. Results: Comparing the findings of our study with the previous anatomical studies, it is found that we studied a large number of sulci in detail. When we compared the anatomical findings with the findings of USG or MRI it was found that there was 2–4 weeks delay between anatomical appearance and visualization of the same sulcus in fetal brain by imaging (USG and MRI). The development of cerebral sulci was proportional to the bodyweight of the fetus. Conclusion: Embryological appearance of sulci is a crucial important in the Anatomy and radiological study of brain and micro neurosurgery. The study is essential for estimation of the gestational age of the fetus also.

Takahata et al. investigate the functional role of SMOC1/2 proteins in skeletal development. They reveal a genetic pathway that includes Bmp2 and Runx2 inducing expression of the paralogous Smoc genes, which may offer novel and effective therapeutic strategies associated with various bone and cartilage diseases.

Questioning what regulates left-right asymmetry breaking in the mouse node: the authors identify a 200 bp stretch of the Dand5 3’UTR where Bicc1 binds, and Cnot proteins downstream of calcium flow regulate the post-transcriptional regulation of Dand5 by Bicc1.

Summary: Uncoupling protein 2 (UCP2), a mitochondrial protein, is known to be upregulated in pancreatic islets of patients with type 2 diabetes (T2DM); however, the pathological significance of this increase in UCP2 expression is unclear. In this study, we highlight the molecular link between the increase in UCP2 expression in β-cells and β-cell failure by using genetically engineered mice and human islets. β-cell-specific UCP2-overexpressing transgenic mice (βUCP2Tg) exhibited glucose intolerance and a reduction in insulin secretion. Decreased mitochondrial function and increased aldolase B (AldB) expression through oxidative-stress-mediated pathway were observed in βUCP2Tg islets. AldB, a glycolytic enzyme, was associated with reduced insulin secretion via mitochondrial dysfunction and impaired calcium release from the endoplasmic reticulum (ER). Taken together, our findings provide a new mechanism of β-cell dysfunction by UCP2 and AldB. Targeting the UCP2/AldB axis is a promising approach for the recovery of β-cell function.

Abstract Background Cardiac pacing is a growing activity in Sub-Saharan Africa. There is little data on the characteristics of this interventional treatment in our regions. The goal was to evaluate the results of cardiac pacing in a referral service in sub-Saharan Africa. Methods We carried out a twelve-year retrospective study (from January 1st, 2004 to December 31st, 2015) in the Cardiology Department of Aristide Le Dantec University Hospital. This work included all patients who received definitive cardiac pacing during the study period and followed up in the service. Results In total we included 606 patients. There was a growing trend in activity with a peak in 2015 (17%). The average age was 70.6 ± 12.03 years. Some patients (15.4%) came from the subregion. The patients were mostly of medium socio-economic level (53%); 14% were of low socio-economic level. Patients were symptomatic in 85% of cases (37.4% syncope). The indications were dominated by complete atrioventricular block (81.5%); sinus dysfunction accounted for 1.9% of them. A temporary pacemaker was used in 60% of cases for an average duration of 5.1 ± 6.3 days. Antibiotics, local anesthesia and analgesics were used in all cases. Implanted pacemakers were single chamber in 56% of cases and double chamber in 44% of cases. In 39 patients (6.4%), the pacemaker was a « re-used » one. The atrial leads were most often placed in a lateral position (94.5%). The ventricular ones were predominantly tined (95.7%) and more often located at the apical level. Complications were noted in 24 patients (3.9%), dominated by devices externalizations and infections, which together accounted for 2.7% of cases. The number of people in the cathlab was significantly higher and the duration of the temporary pacemaker was longer for patients who had a complication. There was no significant difference depending on the type of pacemaker used (new or reused). Seven (7) in hospital death cases were reported. Conclusion Cardiac pacing is a growing activity in Dakar.

Background: Langerhans cells (LCs) polarize the immune milieu towards a T helper type (Th) 1 or Th2 immune response. We investigated the effects of selected tetracyclines on Th cells development mediated by LCs, and their implications for the treatment of atopic dermatitis (AD). Methods: Mice were primed with ovalbumin (OVA) peptide-pulsed LCs, which had been treated with each antibiotic, via the hind footpad. After 5 days, the Th1/Th2 cytokine response in the popliteal lymph nodes was investigated by enzyme-linked immunosorbent assay. The expression of cell surface molecules on LCs was investigated using reverse transcriptase polymerase chain reaction. The therapeutic effects of a selected antibiotic on AD-like skin lesions of NC/Nga mice were assessed in terms of the skin severity score, histological changes in the lesioned skin, the serum level of total IgE, and expression of Th1/Th2 cytokines in lymph nodes and skin lesions. Results: Antibiotic-treated, OVA peptide-pulsed LCs inhibited development of Th2 cells but not Th1 cells. This was accompanied by suppression of T-cell immunoglobulin and mucin domain-containing protein (TIM)-4 expression in LCs. Doxycycline had the greatest activity against Staphylococcus aureus strains isolated from skin lesions of patients with AD, and a strong inhibitory effect on Th2 cell development. Doxycycline suppressed the increase in the skin severity score during the acute phase in NC/Nga mice similar to betamethasone. This suppressive effect was associated with a decrease in the serum IgE level and production of Th2 cytokines in auricular lymph node cells and skin lesions. Conclusion: Topical application of doxycycline to AD lesions would act on both superficial S. aureus colonization and epidermal LCs, thus possibly inhibiting the development of Th2 cells in vivo, with benefits for control of acute inflammation in AD.

Dendrite microtubules are polarized with minus-end-out orientation in Drosophila neurons. Nucleation sites concentrate at dendrite branch points, but how they localize is not known. Using Drosophila, we found that canonical Wnt signaling proteins regulate localization of the core nucleation protein γTubulin (γTub). Reduction of frizzleds (fz), arrow (low-density lipoprotein receptor-related protein [LRP] 5/6), dishevelled (dsh), casein kinase Iγ, G proteins, and Axin reduced γTub-green fluorescent protein (GFP) at branch points, and two functional readouts of dendritic nucleation confirmed a role for Wnt signaling proteins. Both dsh and Axin localized to branch points, with dsh upstream of Axin. Moreover, tethering Axin to mitochondria was sufficient to recruit ectopic γTub-GFP and increase microtubule dynamics in dendrites. At dendrite branch points, Axin and dsh colocalized with early endosomal marker Rab5, and new microtubule growth initiated at puncta marked with fz, dsh, Axin, and Rab5. We propose that in dendrites, canonical Wnt signaling proteins are housed on early endosomes and recruit nucleation sites to branch points.

Abstract Background Cardiovascular complications in Marfan syndrome (MFS) make all its seriousness. Taking as a basis the Ghent criteria, we conducted a family screening from an index case. The objective was to describe the clinical characteristics of MFS anomalies and to detect cardiovascular complications in our patients. Case presentation Six subjects were evaluated. Patients had to be in the same uterine siblings of the index case or be a descendant. The objective was to search for MFS based on the diagnostic criteria of Ghent and, subsequently, detecting cardiovascular damage. The average age was 24 years. The examination revealed three cases of sudden death in a context of chest pain. Five subjects had systemic involvement with a score ≥ 7 that allowed to the diagnosis of MFS. Two patients had simultaneously ectopia lentis and myopia. In terms of cardiovascular damage, there were three cases of dilatation of the aortic root, two cases of aortic dissection of Stanford’s type A with severe aortic regurgitation in one case and moderate in the other. There were three patients with moderate mitral regurgitation with a case by valve prolapse. Conclusion The family screening is crucial in Marfan syndrome. It revealed serious cardiovascular complications including sudden death and aortic dissection.

INTRODUCTION: Le syndrome cardio-rénal (SCR) est un trouble physiopathologique du cèur et des reins dans lequel une dysfonction chronique ou aiguë de l'un peut induire une dysfonction chronique ou aiguë de l'autre. En Afrique et au Sénégal en particulier l'incidence du syndrome cardio-rénal est mal connue. L'objectif de cette étude était d'apprécier la prévalence du SCR en milieu cardiologique. METHODES: Nous avons mené une étude rétrospective incluant tous les insuffisants cardiaques ayant une altération de leur fonction rénale et hospitalisés entre avril 2010 et avril 2011 au service de cardiologie. Les données étaient analysées avec le logiciel statistique Epi-info 3.5.3. RESULTATS: Nous avons inclus 36 patients. La prévalence était de 3,7% avec une prédominance masculine (sex-ratio à 1,77) et un âge moyen à 56,9 ans [30-92]. Les antécédents étaient dominés par l'hypertension artérielle (52,77%) et le diabète (19,4%). Les principales étiologies étaient la cardiomyopathie hypertensive (39%) et l'insuffisance coronarienne (19,44%). La symptomatologie était dominée par la dyspnée (69,4%) et les èdèmes (50%). On notait une anémie (17 patients). La clairance moyenne (MDRD) à 46 ml/min. L'échocardiographie Doppler retrouvait majoritairement des troubles de la cinétique (89,3%), une dysfonction systolique VG (71%). Les 3 échographies rénales étaient normales. Six décès (16,7 %) étaient notés. CONCLUSION: Le syndrome cardio-rénal est une réalité et marque un tournant dans l'évolution de toute cardiopathie et néphropathie. Sa prévalence en milieu cardiologique sénégalais est faible. Des études prospectives et multicentriques sont nécessaires pour une meilleure évaluation au Sénégal.

INTRODUCTION: l'hypertension artérielle (HTA) du sujet âgé est un facteur indépendant de maladie cardio-vasculaire. Nos objectifs étaient de décrire les aspects cliniques, électrocardiographique et échocardiographiques de l'HTA du sujet âgé. METHODES: nous avons mené une étude descriptive et transversale de Janvier à Septembre 2013. Etaient inclus les sujets hypertendus âgés d'au moins 60 ans suivis en ambulatoire au service de cardiologie de l'Hôpital Principal de Dakar. Les données statistiques étaient analysées par le logiciel Epi Info 7 et une valeur de p = 0,05 était retenue comme significative. RESULTATS: au total, 208 patients étaient inclus. L'âge moyen était de 69,9 ans avec une prédominance féminine (sex-ratio de 0,85). La pression artérielle moyenne était de 162/90mmHg. L'HTA était contrôlée dans 13% des cas. A l'électrocardiogramme, on notait un trouble du rythme (17,78%), une hypertrophie auriculaire gauche (45,19%), une hypertrophie ventriculaire gauche (28,85%) et 2 cas de bloc auriculo-ventriculaire complet. Le Holter ECG révélait 4 cas de tachycardie ventriculaire non soutenue (IVb de Lown), 6 cas de fibrillation atriale paroxystique et 1 cas de flutter atrial paroxystique. L'échocardiographie réalisée chez 140 patients retrouvait une HVG à prédominance concentrique chez 25 patients, plus fréquente chez les hommes (p=0,04) et une dilatation de l' gauche dans 56,42% des cas, plus fréquente chez les patients plus âgés (p= 0,01). CONCLUSION: les aspects électrocardiographiques et échocardiographiques dans la population hypertendue âgée sont caractérisés par l'hypertrophie ventriculaire gauche notamment concentrique, la fréquence des arythmies révélées quelques fois par l'enregistrement électrocardiographique de longue durée.

The agouti signaling protein (ASIP)/melanocortin signaling system regulates lipid metabolism in humans but not in mice. To examine whether the system is present in adipose tissues in chickens, we performed reverse transcription-polymerase chain reaction (RT-PCR) analysis using mRNAs extracted from subcutaneous and visceral adipose tissues. This revealed that mRNAs for melanocortin 1 receptor (MC1R), MC5R, ASIP, pro-opiomelanocortin (POMC), prohormone convertase 1 (PC1) and PC2 were expressed in both adipose tissues, suggesting the presence of a local ASIP/melanocortin signaling system in these tissues in chickens. To investigate the potential biological role of this system, the adipose tissue mRNA expression of ASIP and POMC was examined under food-deprived conditions. Forty-eight-hour fasting significantly decreased body weight and reduced the cell size in adipose tissues. In addition, the plasma levels of free fatty acids were increased while those of glucose, triacylglycerols and insulin were decreased, suggesting the activation of lipolysis and gluconeogenesis in the fasted chickens. Under this condition, the mRNA expression of ASIP and POMC was decreased and increased, respectively, in subcutaneous and visceral adipose tissues. ASIP and melanocortins are lipogenic and lipolytic hormones, respectively. Therefore, these findings suggest that lipid metabolism in the adipose tissues is regulated by a local ASIP/melanocortin signaling system in chickens. This is the first report demonstrating changes in the expression of ASIP mRNA in adipose tissues in the negative-energy state in vertebrates.

Diphenyldiazomethane with four iodine groups at the ortho positions and two tert-butyl groups at the para positions, i.e., bis(4-tert-butyl-2,6-diiodophenyl)diazomethane (1a-N2), was synthesized as a sterically hindered triplet carbene precursor. Irradiation of 1a-N2 in solution effectively generated the corresponding triplet diphenylcarbene 31a, which was characterized by UV-vis spectroscopy at low temperature, along with laser flash photolysis techniques at room temperature. The UV-vis spectrum of 31a was obtained by irradiating 1a-N2 in a 2-methyltetrahydrofuran matrix at 77 K. The ESR spectrum showed no triplet carbene signals, while a radical species was observed at the anticipated temperature of the decomposition of triplet carbene 31a. Transient absorption bands ascribable to 31a were observed by laser flash photolysis of 1a-N2 in a degassed benzene solution and decayed very slowly with a second-order rate constant (2k/εl) of 5.5 × 10−3·s−1. Steady-state irradiation of 1a-N2 in degassed benzene afforded 9,10-diarylphenanthrene derivative 2a in a 31% yield. Triplet carbene 31a was also trapped by either oxygen (kO2 = 6.5 × 105 M−1·s−1) or 1,4-cyclohexadiene (kCHD = 1.5 M−1·s−1) to afford the corresponding ketone 1a-O or the diarylmethane 1a-H2. The carbene was shown to be much less reactive than the triplet diphenylcarbene that is protected by two ortho-iodo and two ortho-bromo groups, 31b.

Abstract Background Mycobacterium tuberculosis can enter into a dormant state which has resulted in one third of the world's population being infected with latent tuberculosis making the study of latency and reactivation of utmost importance. M. tuberculosis encodes five resuscitation promoting factors (Rpfs) that bear strong similarity to a lysozyme-like enzyme previously implicated in reactivation of dormant bacteria in vitro. We have developed an intraperitoneal infection model in mice, with immune modulation, that models chronic infection with similar properties in mouse lungs as those observed in the murine aerosol infection model. We have assessed the behavior of mutants that lack two or three rpf genes in different combinations in our intraperitoneal model. Methods C57Bl/6 mice were intraperitonealy infected with H37Rv wild type M. tuberculosis or mutant strains that lacked two or three rpf genes in different combinations. After 90 days of infection aminoguanidine (AG) or anti-TNFα antibodies were administrated. Organ bacillary loads were determined at various intervals post infection by plating serial dilutions of organ homogenates and enumerating bacteria. Results We found that the rpf triple and double mutants tested were attenuated in their ability to disseminate to mouse lungs after intraperitoneal administration and were defective in their ability to re-grow after immunosuppression induced by administration of aminoguanidine and anti-TNFα antibodies. Conclusion Rpf proteins may have a significant physiological role for development of chronic TB infection and its reactivation in vivo.

Ashwin and Chanagan are double-stranded siphoviral mycobacteriophages isolated from a single soil sample from a lavender flowerpot in Johannesburg, South Africa. Both are capable of infecting and lyzing Mycobacterium smegmatis and are predicted to contain 122 and 91 open reading frames, respectively, most without known function.

Lopsy is a siphovirus mycobacteriophage that is capable of lytic infection in Mycobacterium smegmatis. It is classified as a subcluster B1 mycobacteriophage and was isolated from soil in Estcourt, South Africa. The 68,542-bp double-stranded DNA genome is circularly permuted, has a GC content of 66.4%, and is predicted to contain 98 genes., Competing Interests: The authors declare no conflict of interest.

The mucosal environment of the upper respiratory tract is the first barrier of protection against SARS-CoV-2 transmission. However, the mucosal factors involved in viral transmission and potentially modulating the capacity to prevent such transmission have not fully been identified. In this pilot proteomics study, we compared mucosal and systemic compartments in a South African cohort of vaccinated and unvaccinated individuals undergoing maxillofacial surgery with previous history of COVID-19 or not. Inflammatory profiles were analyzed in plasma, nasopharyngeal swabs, and nasal and oral tissue explant cultures, using Olink and Luminex technologies. SARS-CoV-2-specific antibody levels were measured in serum and tissue explants. An increased pro-inflammatory proteomic profile was measured in the nasal compartment compared to plasma. However, IP-10 and MIG levels were higher in secretions than in nasal tissue, and the opposite was observed for TGF-β. Nasal anti-SARS-CoV-2 spike IgG correlated with mucosal MIG expression for all participants. A further positive correlation was found with IP-10 in BioNTech/Pfizer-vaccinated individuals. Systemic levels of anti-SARS-CoV-2 spike IgG elicited by this vaccine correlated with plasma IL-10, IL-6 and HBD4. Proteomic profiles measured in mucosal tissues and secretions using combined technologies could reveal correlates of protection at the mucosal portals of viral entry.

Tuberculosis (TB) infected individuals harbor a heterogenous population of differentially culturable tubercle bacilli (DCTB). Herein, we describe how DCTB assays using culture filtrate either containing or deficient in resuscitation promoting factors can uncover mixed infections. We demonstrate that Mycobacterium tuberculosis ( Mtb ) strain genotypes can be separated in DCTB assays based on their selective requirement for growth stimulatory factors. Beijing mixed infections appear to be associated with a higher bacterial load and reduced reliance on growth stimulatory factors. These data have important implications for identifying mixed infections and hetero-resistance, which in turn can affect selection of treatment regimen and establishment of transmission links., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Chengalroyen, Beukes, Otwombe, Gordhan, Martinson and Kana.)

IgnatiusPatJac is a Siphoviridae mycobacteriophage capable of lytic infection in Mycobacterium smegmatis and Mycobacterium tuberculosis. It was isolated from damp soil in Johannesburg, South Africa. The 51,164-bp double-stranded DNA genome has a GC content of 63.6%, predicted to encode 93 genes. IgnatiusPatJac is classified as an A1 subcluster mycobacteriophage.

Peptidoglycan (PG) is the exoskeleton of bacterial cells and is required for their viability, growth, and cell division. Unlike most bacteria, mycobacteria possess an atypical PG characterized by a high degree of unique linkages and chemical modifications which most likely serve as important determinants of virulence and pathogenesis in mycobacterial diseases. Despite this important role, the chemical composition and molecular architecture of mycobacterial PG have yet to be fully determined. Here we determined the chemical composition of PG from Mycobacterium smegmatis using high-resolution liquid chromatography-mass spectrometry. Purified cell walls from the stationary phase were digested with mutanolysin and compositional analysis was performed on 130 muropeptide ions that were identified using an in silico PG library. The relative abundance for each muropeptide ion was measured by integrating the extracted-ion chromatogram. The percentage of crosslink per PG subunit was measured at 45%. While both 3→3 and 4→3 transpeptide cross-linkages were found in PG dimers, a high abundance of 3→3 linkages was found associated with the trimers. Approximately 43% of disaccharides in the PG of M. smegmatis showed modifications by acetylation or deacetylation. A significant number of PG trimers are found with a loss of 41.00 amu that is consistent with N-deacetylation, whereas the dimers show a gain of 42.01 amu corresponding to O-acetylation of the PG disaccharides. This suggests a possible role of PG acetylation in the regulation of cell wall homeostasis in M. smegmatis. Collectively, these data report important novel insights into the ultrastructure of mycobacterial PG., (© 2022. The Author(s).)

Peptidoglycan (PG), a heteropolysaccharide component of the mycobacterial cell wall can be shed during tuberculosis infection with immunomodulatory consequences. As such, changes in PG structure are expected to have important implications on disease progression and host responses during infection with Mycobacterium tuberculosis . Mycobacterial amidases have important roles in remodeling of PG during cell division and are implicated in susceptibility to antibiotics. However, their role in modulating host immunity remains unknown. We assessed the bacterial burden and host immune responses to M. tuberculosis mutants defective for either one of two PG N-acetylmuramyl-L-alanine amidases, Ami1 and Ami4, in bone marrow-derived macrophages (BMDM) and C57BL/6 mice. In infected BMDM, the single deletion of both genes resulted in increased proinflammatory cytokine responses. In mice, infection with the Δ ami1 mutant led to differential induction of pro-inflammatory cytokines and chemokines, decreased cellular recruitment and reduced lung pathology during the acute phase of the infection. While increased proinflammatory cytokines production was observed in BMDM infected with the Δ ami4 mutant, these effects did not prevail in mice. Infection using the Δ ami1 and Δ ami4 Mtb mutants showed that these genes are dispensable for intracellular mycobacterial growth in macrophages and mycobacterial burden in mice. These findings suggest that both Ami1 and Ami4 in M. tuberculosis are not essential for mycobacterial growth within the host. In summary, we show that amidases are important for modulating host immunity during Mtb infection in murine macrophages and mice.

There is an urgent need for point-of-care tuberculosis (TB) diagnostic methods that are fast, inexpensive, and operationally simple. Here, we report on a bright solvatochromic dye trehalose conjugate that specifically detects Mycobacterium tuberculosis (Mtb) in minutes. 3-Hydroxychromone (3HC) dyes, known for having high fluorescence quantum yields, exhibit shifts in fluorescence intensity in response to changes in environmental polarity. We synthesized two analogs of 3HC-trehalose conjugates (3HC-2-Tre and 3HC-3-Tre) and determined that 3HC-3-Tre has exceptionally favorable properties for Mtb detection. 3HC-3-Tre-labeled mycobacterial cells displayed a 10-fold increase in fluorescence intensity compared to our previous reports on the dye 4,4- N,N -dimethylaminonapthalimide (DMN-Tre). Excitingly, we detected fluorescent Mtb cells within 10 min of probe treatment. Thus, 3HC-3-Tre permits rapid visualization of mycobacteria that ultimately could empower improved Mtb detection at the point-of-care in low-resource settings., Competing Interests: The authors declare the following competing financial interest(s): M.K. and C.R.B. are cofounders of OliLux Biosciences which has licensed patents related to the work in this paper. B.K. is a scientific advisor to OliLux Biosciences. B.K is cofounder of SmartSpot Quality CC and scientific advisor to SmartSpot Quality CC and Pulmosat. C.R.B. is a cofounder and scientific advisory board member of Lycia Therapeutics, Palleon Pharmaceuticals, Enable Bioscience, Redwood Biosciences (a subsidiary of Catalent), and InterVenn Biosciences, and a member of the board of directors of Eli Lilly & Company., (© 2021 The Authors. Published by American Chemical Society.)

Diagnosis of tuberculosis in pediatric patients remains challenging due to inherent difficulties associated with obtaining respiratory samples for molecular and culture-based testing. To address this, recent studies have highlighted the utility of tongue swabs to detect Mycobacterium tuberculosis genomic DNA in the oral epithelia of tuberculosis infected adults. It is unknown whether tongue swabs have similar utility for diagnosis of childhood tuberculosis and if the presence of DNA in these swabs was associated with whole bacilli. We therefore sought to conduct a preliminary assessment of the utility of tongue swabs to detect tubercle bacilli and their associated genetic material in young children. For this, we recruited hospitalized children with clinically diagnosed tuberculosis ( n = 26) or lower respiratory tract infection (LRTI, n = 9). These categories were blinded for downstream laboratory tests, which included PCR, spoligotyping, smear microscopy, and culture. Mtb genomic DNA was detected by PCR only in clinically diagnosed TB cases [11/26 (31.4%)] and not in cases with LRTI. Of these, 5/11 [45.5%] were associated with a spoligotype. Spoligotyping also detected an additional six specimens that were negative by PCR. Using smear microscopy, 19/26 [73.1%] and 4/9 [44.4] were Mtb positive in the tuberculosis or LRTI categories respectively. We noted positive results on all three tests in 5/26 [19.2%] in the tuberculosis category and 0/9 in the LRTI category. All specimens were culture negative. Collectively, these preliminary data present a compelling case for broader testing of tongue swabs to diagnose tuberculosis in children where obtaining standard sputum specimens is not easy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ealand, Peters, Jacobs, Sewcharran, Ghoor, Golub, Brahmbhatt, Martinson, Dangor, Lala and Kana.)

Background: Pulmonary tuberculosis (TB) in people living with HIV (PLH) frequently presents as sputum smear-negative. However, clinical trials of TB in adults often use smear-positive individuals to ensure measurable bacterial responses following initiation of treatment, thereby excluding HIV-infected patients from trials., Methods: In this prospective case cohort study, 118 HIV-seropositive TB patients were assessed prior to initiation of standard four-drug TB therapy and at several time points through 35 days. Sputum bacillary load, as a marker of treatment response, was determined serially by: smear microscopy, Xpert MTB/RIF, liquid culture, and colony counts on agar medium., Results: By all four measures, patients who were baseline smear-positive had higher bacterial loads than those presenting as smear-negative, until day 35. However, most smear-negative PLH had significant bacillary load at enrolment and their mycobacteria were cleared more rapidly than smear-positive patients. Smear-negative patients' decline in bacillary load, determined by colony counts, was linear to day 7 suggesting measurable bactericidal activity. Moreover, the decrease in bacterial counts was comparable to smear-positive individuals. Increasing cycle threshold values (Ct) on the Xpert assay in smear-positive patients to day 14 implied decreasing bacterial load., Conclusion: Our data suggest that smear-negative PLH can be included in clinical trials of novel treatment regimens as they contain sufficient viable bacteria, but allowances for late exclusions would have to be made in sample size estimations. We also show that increases in Ct in smear-positive patients to day 14 reflect treatment responses and the Xpert MTB/RIF assay could be used as biomarker for early treatment response.

Objectives: Describe novel methods for ascertaining verbal fluency in a large national sample of adults, examine demographic factors influencing performance, and compare scores to studies using in-person assessment., Methods/design: Participants were from the Reasons for Geographic and Racial Differences in Stroke (REGARDS) study, a national, population-based, longitudinal study of stroke in adults aged 45 years and older. Letter and semantic fluency were gathered, using Letter "F" and Animal Naming, via a telephone-based assessment with computer-assisted scoring of digital recordings., Results: Initial letter and semantic fluency scores were obtained on 18 505 and 18 072 participants, respectively. For both fluency tests, scores were normally distributed. Younger age and more years of education were associated with better performances (p < 0.0001). The mean and standard deviation for matched subgroups, based on age, gender, and education, were quite comparable with scores reported out of samples using an in-person administration format. Telephone-based assessment also allowed for a level of quality control not available via in-person measurement., Conclusions: Telephone-based assessment of verbal fluency and computer-assisted scoring programs designed for this study facilitated large-scale data acquisition, storage, and scoring of protocols. The resulting scores have similar characteristics to those obtained by traditional methods. These findings extend validation of cognitive assessment methods, using survey research staff and computer-assisted technology for test administration., (© 2018 John Wiley & Sons, Ltd.)

Resuscitation-promoting factors (Rpfs) have previously been shown to act as growth-stimulatory molecules via their lysozyme-like activity on peptidoglycan in the bacterial cell wall. In this study, we investigated the ability of Mycobacterium smegmatis strains lacking rpf genes to form biofilms and tested their susceptibilities to cell wall-targeting agents. M. smegmatis contains four distinct rpf homologues, namely, MSMEG&#95;5700 ( rpfA ), MSMEG&#95;5439 ( rpfB ), MSMEG&#95;4640 ( rpfE2 ), and MSMEG&#95;4643 ( rpfE ). During axenic growth of the wild-type strain, all four mRNA transcripts were expressed to various degrees, but the expression of MSMEG&#95;4643 was significantly greater during exponential growth. Similarly, all rpf mRNA transcripts could be detected in biofilms grown for 7, 14, and 28 days, with MSMEG&#95;4643 expressed at the highest abundance after 7 days. In-frame unmarked deletion mutants (single and combinatorial) were generated and displayed altered colony morphologies and the inability to form typical biofilms. Moreover, any strain lacking rpfA and rpfB simultaneously exhibited increased susceptibility to rifampin, vancomycin, and SDS. Exogenous Rpf supplementation in the form of culture filtrate failed to restore biofilm formation. Liquid chromatography-mass spectrometry (LC-MS) analysis of peptidoglycan (PG) suggested a reduction in 4-3 cross-linked PG in the Δ rpfABEE2 mutant strain. In addition, the level of PG-repeat units terminating in 1,6-anhydroMurNAc appeared to be significantly reduced in the quadruple rpf mutant. Collectively, our data have shown that Rpfs play an important role in biofilm formation, possibly through alterations in PG cross-linking and the production of signaling molecules. IMPORTANCE The cell wall of pathogenic mycobacteria is composed of peptidoglycan, arabinogalactan, mycolic acids, and an outer capsule. This inherent complexity renders it resistant to many antibiotics. Consequently, its biosynthesis and remodeling during growth directly impact viability. Resuscitation-promoting factors (Rpfs), enzymes with lytic transglycosylase activity, have been associated with the revival of dormant cells and subsequent resumption of vegetative growth. Mycobacterium smegmatis , a soil saprophyte and close relative of the human pathogen Mycobacterium tuberculosis , encodes four distinct Rpfs. Herein, we assessed the relationship between Rpfs and biofilm formation, which is used as a model to study drug tolerance and bacterial signaling in mycobacteria. We demonstrated that progressive deletion of rpf genes hampered the development of biofilms and reduced drug tolerance. These effects were accompanied by a reduction in muropeptide production and altered peptidoglycan cross-linking. Collectively, these observations point to an important role for Rpfs in mycobacterial communication and drug tolerance., (Copyright © 2018 American Society for Microbiology.)

Mycobacteria possess a multi-layered cell wall that requires extensive remodelling during cell division. We investigated the role of an amidase&#95;3 domain-containing N-acetylmuramyl-L-alanine amidase, a peptidoglycan remodelling enzyme implicated in cell division. We demonstrated that deletion of MSMEG&#95;6281 (Ami1) in Mycobacterium smegmatis resulted in the formation of cellular chains, illustrative of cells that were unable to complete division. Suprisingly, viability in the Δami1 mutant was maintained through atypical lateral branching, the products of which proceeded to form viable daughter cells. We showed that these lateral buds resulted from mislocalization of DivIVA, a major determinant in facilitating polar elongation in mycobacterial cells. Failure of Δami1 mutant cells to separate also led to dysregulation of FtsZ ring bundling. Loss of Ami1 resulted in defects in septal peptidoglycan turnover with release of excess cell wall material from the septum or newly born cell poles. We noted signficant accumulation of 3-3 crosslinked muropeptides in the Δami1 mutant. We further demonstrated that deletion of ami1 leads to increased cell wall permeability and enhanced susceptiblity to cell wall targeting antibiotics. Collectively, these data provide novel insight on cell division in actinobacteria and highlights a new class of potential drug targets for mycobacterial diseases.

Background: HIV-uninfected individuals with pulmonary TB (PTB) commonly present with radiological features of upper lobe cavitatory disease. In contrast, individuals with HIV and PTB may present differently. This study compared radiological features of individuals with laboratory-confirmed PTB by HIV status from the largest study in South Africa., Methods: We conducted a cross-sectional analysis of adults recruited between 2012 and 2015 with laboratory-confirmed PTB in Soweto, South Africa. Baseline characteristics and chest radiograph (CXR) findings were compared by χ2 test stratified by HIV status., Results: Of the 474 individuals with PTB, 348 (73.4%) had HIV. Individuals with HIV had a higher proportion of infiltrates (58.9% vs 46.8%, p=0.02) and a lower proportion of cavitations (40.8% vs 68.3%; p<0.0001) compared to HIV-uninfected individuals. Additionally, individuals with HIV had a lower proportion of cavitations sized ≥4 cm (16.7% vs 36.5%, p<0.001) and a lower proportion of disease extent involving half or more of the total lung area radiologically (25.9% vs 45.3%, p<0.0001)., Conclusions: Individuals with HIV co-infected with PTB have a higher proportion of infiltrates and a lower proportion of cavitations relative to the HIV-uninfected PTB individuals. The absence of classical upper lobe cavitatory disease on CXR does not exclude PTB in individuals with HIV., (© The Author 2017. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)