Actin-capping protein regulates actomyosin contractility to maintain germline architecture in C. elegans.

Actin dynamics play an important role in tissue morphogenesis, yet the control of actin filament growth takes place at the molecular level. A challenge in the field is to link the molecular function of actin regulators with their physiological function. Here, we report an in vivo role of the actin-capping protein CAP-1 in the Caenorhabditis elegans germline. We show that CAP-1 is associated with actomyosin structures in the cortex and rachis, and its depletion or overexpression led to severe structural defects in the syncytial germline and oocytes. A 60% reduction in the level of CAP-1 caused a twofold increase in F-actin and non-muscle myosin II activity, and laser incision experiments revealed an increase in rachis contractility. Cytosim simulations pointed to increased myosin as the main driver of increased contractility following loss of actin-capping protein. Double depletion of CAP-1 and myosin or Rho kinase demonstrated that the rachis architecture defects associated with CAP-1 depletion require contractility of the rachis actomyosin corset. Thus, we uncovered a physiological role for actin-capping protein in regulating actomyosin contractility to maintain reproductive tissue architecture.

Pathway to Independence: the future of developmental biology.

In 2022, Development launched its Pathway to Independence (PI) Programme, aimed at supporting postdocs as they transition to their first independent position. We selected eight talented researchers as the first cohort of PI Fellows. In this article, each of our Fellows provides their perspective on the future of their field. Together, they paint an exciting picture of the current state of and open questions in developmental biology.

Active nuclear positioning and actomyosin contractility maintain leader cell integrity during gonadogenesis.

Proper distribution of organelles can play an important role in a moving cell's performance. During C. elegans gonad morphogenesis, the nucleus of the leading distal tip cell (DTC) is always found at the front, yet the significance of this localization is unknown. Here, we identified the molecular mechanism that keeps the nucleus at the front, despite a frictional force that pushes it backward. The Klarsicht/ANC-1/Syne homology (KASH) domain protein UNC-83 links the nucleus to the motor protein kinesin-1 that moves along a polarized acentrosomal microtubule network. Interestingly, disrupting nuclear positioning on its own did not affect gonad morphogenesis. However, reducing actomyosin contractility on top of nuclear mispositioning led to a dramatic phenotype: DTC splitting and gonad bifurcation. Long-term live imaging of the double knockdown revealed that, while the gonad attempted to perform a planned U-turn, the DTC was stretched due to the lagging nucleus until it fragmented into a nucleated cell and an enucleated cytoplast, each leading an independent gonadal arm. Remarkably, the enucleated cytoplast had polarity and invaded, but it could only temporarily support germ cell proliferation. Based on a qualitative biophysical model, we conclude that the leader cell employs two complementary mechanical approaches to preserve its integrity and ensure proper organ morphogenesis while navigating through a complex 3D environment: active nuclear positioning by microtubule motors and actomyosin-driven cortical contractility.

Christie, Atkins, Munch-Peterson (CAMP) Negative Listeria monocytogenes Meningitis in Neonates: Report of Two Cases.

Listeria monocytogenes, a gram-positive bacillus and an intracellular pathogen, is an uncommon cause of illness in the general population. During pregnancy, a perinatal infection can lead to serious complications such as abortion, stillbirth, neonatal sepsis, and meningitis. We present two cases of neonatal meningitis caused by Christie, Atkins, Munch-Peterson (CAMP)-negative Listeria monocytogenes. In the first case, a seven-day-old female term neonate delivered vaginally, presented with high-grade fever and refusal to feed. In view of the suspected late-onset sepsis, a septic workup, including cerebrospinal fluid analysis, was conducted. CSF culture reports obtained showed a growth consistent with Listeria monocytogenes, which was CAMP test negative and susceptible to the penicillin group of drugs, cotrimoxazole, erythromycin, and meropenem. The isolate was identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and confirmed by 16S rRNA sequencing. The blood culture was sterile. At 48 hours of admission, the neonate clinically deteriorated with fluctuation in oxygen saturation below 95% at room air. Thus, she was electively intubated and connected to the mechanical ventilator with appropriate settings. The antibiotics were upgraded to meropenem from the empirical antibiotic therapy. The neonate showed clinical improvement within the next 24 hours of initiating antibiotics according to culture susceptibility and was gradually weaned from the mechanical ventilator to continuous positive airway pressure (CPAP). After 24 hours, she was able to maintain normal saturation at room air. In the second case, an 11-day-old low birth weight neonate, small for gestational age, was presented to the NICU with complaints of loose stools, fever, and refusal to feed for the past two days. In view of the suspected sepsis, relevant investigations were carried out while initiating empirical antibiotics IV piperacillin-tazobactam and IV amikacin for the neonate. Meanwhile, there was a dip in oxygen saturation noted on room air for the neonate and he/she was mechanically ventilated. The CSF culture grew Listeria monocytogenes,which was identified using MALDI-TOF MS and confirmed by 16S rRNA sequencing. The isolate tested negative for the CAMP test and was susceptible to ampicillin, penicillin, cotrimoxazole, erythromycin, and meropenem. The blood culture was sterile. The antibiotics were upgraded to meropenem from the empirical antibiotic therapy, the patient's condition improved, and the baby was eventually discharged.

Directed cell invasion and asymmetric adhesion drive tissue elongation and turning in C. elegans gonad morphogenesis.

Development of the C. elegans gonad has long been studied as a model of organogenesis driven by collective cell migration. A somatic cell named the distal tip cell (DTC) is thought to serve as the leader of following germ cells; yet, the mechanism for DTC propulsion and maneuvering remains elusive. Here, we demonstrate that the DTC is not self-propelled but rather is pushed by the proliferating germ cells. Proliferative pressure pushes the DTC forward, against the resistance of the basement membrane in front. The DTC locally secretes metalloproteases that degrade the impeding membrane, resulting in gonad elongation. Turning of the gonad is achieved by polarized DTC-matrix adhesions. The asymmetrical traction results in a bending moment on the DTC. Src and Cdc42 regulate integrin adhesion polarity, whereas an external netrin signal determines DTC orientation. Our findings challenge the current view of DTC migration and offer a distinct framework to understand organogenesis.

Mechanosensing in embryogenesis.

Mechanical forces generated by living cells at the molecular level propagate to the cellular and organismal level and have profound consequences for embryogenesis. A direct result of force application is movement, as occurs in chromosome separation, cell migration, or tissue folding. A less direct, but equally important effect of force, is the activation of mechanosensitive signaling, which allows cells to probe their mechanical surrounding and communicate with each other over short and long distances. In this review, we focus on forces as a means of conveying information and affecting cell behavior during embryogenesis. We discuss four developmental processes that demonstrate the involvement of force in cell fate determination, growth, morphogenesis, and organogenesis, in a variety of model organisms. Finally, a generalizable pathway of mechanosensing and mechanotransduction in vivo is described, and we highlight similarities between morphogens and forces in patterning of embryos.

Validation of Pefloxacin for detection of fluoroquinolone (FQ) resistance among Salmonella Typhi with special reference to GyrB mutations.

Introduction. Fluoroquinolone (FQ) resistant Salmonella are classified as high priority pathogens by WHO. FQ resistance among Salmonella Typhi has emerged rapidly and is predominantly mediated by mutations in the topoisomerase genes gyrA, and parC. Mutations in GyrA result in classical FQ resistance (DCS-NAR) i.e. decreased susceptibility to ciprofloxacin (MIC of 0.12 to 0.5 µg ml-1) (DCS) and resistance to nalidixic acid (NAR). Previously a nalidixic acid disc test was proposed for detection of DCS. Recently isolates with non-classical FQ resistance caused by plasmid-mediated quinolone resistance (PMQR) and mutations in GyrB have emerged. These mechanisms also result in DCS but are nalidixic acid susceptible (NAS) and thus pose diagnostic challenges. CLSI and EUCAST have recommended use of 5 µg pefloxacin discs for detection of DCS in Salmonella.Hypothesis. The CLSI and EUCAST recommendations for use of 5 µg pefloxacin for detection of DCS has not been validated on typhoidal Salmonella and resistance mediated by GyrB mutation in Salmonella species.Aim. The aim of the present study was to validate the performance of the 5 µg pefloxacin discs to detect isolates of S. Typhi with DCS with special reference to GyrB mutations.Methodology. A total of 180 clinical isolates of Salmonella Typhi (2005-2014) were investigated for genetic mechanisms of resistance. Zone diameters for nalidixic acid (30µg), ciprofloxacin (5µg) and pefloxacin (5µg) and minimum inhibitory concentration (MIC) for ciprofloxacin were determined using CLSI guidelines. Performance of the three discs was evaluated to detect FQ resistance in S. Typhi.Results. Topoisomerase mutations in GyrB +/ ParC and GyrB were detected in 112 and 34 isolates respectively. Different mutations have a varied effect on the MIC for ciprofloxacin. The current breakpoints for susceptible (≤0.06 µg ml-1) and non-susceptible (≥0.125 µg ml-1), failed to detect all isolates with a resistance mechanism. Performance of both ciprofloxacin and pefloxacin discs were excellent compared to nalidixic acid in differentiating isolates with non-classical resistance mediated by GyrB from wild-type.Conclusion. The pefloxacin disc can be used to detect FQ resistance among S. Typhi. This is the first report of validation of pefloxacin for detection of FQ resistance in S. Typhi mediated by GyrB mutation.

Investigation of Burkholderia cepacia complex bacteremia outbreak in a neonatal intensive care unit: a case series.

INTRODUCTION: Burkholderia cepacia complex is a ubiquitous organism with a high virulence potential. It is found most commonly in moist environments. Hospital outbreaks have been reported from diverse sources such as contaminated faucets, nebulizers, disinfectant solutions, multidose antibiotic vials, tap water, bottled water, nasal sprays, and ultrasound gels. In this article, we present our experience in investigating and successfully managing an outbreak of nosocomial transmission of Burkholderia cepacia sepsis in the neonatal intensive care unit at SGT Hospital, Haryana, India. CASE PRESENTATION: During the month of March, multiple Burkholderia cepacia complex isolates were recovered from blood cultures of Caucasian babies admitted to the neonatal intensive care unit of our hospital. The organisms were multidrug-resistant, with in vitro sensitivity to meropenem alone (minimum inhibitory concentration = 4 µg/ml). An outbreak was suspected, and the neonatal intensive care unit in-charge and hospital infection control teams were alerted. Outbreak investigation was initiated, and surveillance samples were collected. Burkholderia cepacia complex was successfully isolated from suction apparatus. The isolates were phenotypically typed (biotyping and antimicrobial susceptibility testing) and found to be identical. CONCLUSIONS: In our study, the index case might have been exposed to infection due to a physiological state of low immunity (preterm, low birth weight, and mechanical ventilation). The rest of the cases might have been exposed to this organism due to inadequate hand hygiene/improper cleaning and disinfection practices. Timely reporting and implementation of infection control measures played a significant role in curtailing this outbreak.

Diverse roles of non-muscle myosin II contractility in 3D cell migration.

All is flux, nothing stays still. Heraclitus of Ephesus' characterization of the universe holds true for cells within animals and for proteins within cells. In this review, we examine the dynamics of actin and non-muscle myosin II within cells, and how their dynamics power the movement of cells within tissues. The 3D environment that migrating cells encounter along their path also changes over time, and cells can adopt various mechanisms of motility, depending on the topography, mechanics and chemical composition of their surroundings. We describe the differential spatio-temporal regulation of actin and myosin II-mediated contractility in mesenchymal, lobopodial, amoeboid, and swimming modes of cell migration. After briefly reviewing the biochemistry of myosin II, we discuss the role actomyosin contractility plays in the switch between modes of 3D migration that cells use to adapt to changing environments.

Principles of Actomyosin Regulation In Vivo.

The actomyosin cytoskeleton is responsible for most force-driven processes in cells and tissues. How it assembles into the necessary structures at the right time and place is an important question. Here, we focus on molecular mechanisms of actomyosin regulation recently elucidated in animal models, and highlight several common principles that emerge. The architecture of the actomyosin network - an important determinant of its function - results from actin polymerization, crosslinking and turnover, localized myosin activation, and contractility-driven self-organization. Spatiotemporal regulation is achieved by tissue-specific expression and subcellular localization of Rho GTPase regulators. Subcellular anchor points of actomyosin structures control the outcome of their contraction, and molecular feedback mechanisms dictate whether they are transient, cyclic, or persistent.

Resident Memory and Recirculating Memory T Cells Cooperate to Maintain Disease in a Mouse Model of Vitiligo.

Tissue resident memory T cells (Trm) form in the skin in vitiligo and persist to maintain disease, as white spots often recur rapidly after discontinuing therapy. We and others have recently described melanocyte-specific autoreactive Trm in vitiligo lesions. Here, we characterize the functional relationship between Trm and recirculating memory T cells (Tcm) in our vitiligo mouse model. We found that both Trm and Tcm sensed autoantigen in the skin long after stabilization of disease, producing IFN-γ, CXCL9, and CXCL10. Blockade of Tcm recruitment to the skin with FTY720 or depletion of Tcm with low-dose Thy1.1 antibody reversed disease, indicating that Trm cooperate with Tcm to maintain disease. Taken together, our data provide characterization of skin memory T cells in vitiligo, demonstrate that Trm and Tcm work together during disease, and indicate that targeting their survival or function may provide novel, durable treatment options for patients.

Antiemetic prophylaxis with temozolomide: an audit from a tertiary care center.

BACKGROUND: In our previous experience, a significant proportion of patients who received 5-HT3 antagonist monotherapy with adjuvant temozolomide (150-200 mg/m2) had chemotherapy-induced nausea and vomiting (CINV). This is an audit comparing the multiple antiemetic therapies in the prevention of temozolomide-associated CINV. METHODS: This was a retrospective audit. Adult glioma patients treated with temozolomide at a dose of 150-200 mg/m2 between October 2017 and June 2018 were selected for this analysis. Three antiemetic prophylaxis were used in this time period: ondansetron (October 2017 to November 2017), ondansetron + domperidone (December 2017 to February 2018), and ondansetron + olanzapine (March 2018 to June 2018). The rates of nausea and vomiting were compared among the 3 cohorts using the chi-squared test with Bonferroni correction. A P value of less than .016 was considered significant. RESULTS: A total of 360 patients were selected for this analysis. There were 91 patients in the ondansetron prophylaxis group (25.3%), 113 (31.4%) in the ondansetron plus domperidone group, and 156 (43.3%) in the ondansetron plus olanzapine group. The overall incidence of nausea and vomiting was 25.0% (n = 90) and 7.2% (n = 26). Overall the rates of nausea (P = .052) and vomiting (P = .481) were similar in all 3 cohorts. However, the rates of grade 2 and above nausea (P = .012) and vomiting (P = .015) were significantly lower in the olanzapine group. CONCLUSION: The combination of ondansetron with olanzapine leads to a statistically significant decrease in the rate of moderate-to-severe emesis and nausea and needs to be explored in a prospective study.

Molecular characterisation of Staphylococcus aureus using spa typing as a diagnostic tool in Haryana, India.

BACKGROUND: Staphylococcus aureus is one of the top six most common etiologic agents of nosocomial, community and livestock acquired bacterial infections. These infections although initially were described as a major problem in hospitals have now also become a serious threat in community not only in India but also worldwide. Its prevalence varies depending on the health-care setting, country or a particular region. Thus to better understand the epidemiology of methicillin-resistant S. aureus (MRSA) in a particular geographical location, it is important to study the variations in the population using molecular tools. METHODS: This prospective study was carried out in the Department of Microbiology of Shree Guru Gobind Singh Tricentenary (SGT) Medical College. Staphylococcal protein A (spa) typing was done on 250 S. aureus isolates obtained from various clinical specimens including pus, wound swabs, urine, catheters, blood and cerebrosspinal fluid from both indoor and outdoor patients of SGT Hospital, Budhera, Gurgaon. RESULTS: The selected region of the spa gene of all 250 isolates which includes 87 MRSA and 163 methicillin-susceptible S. aureus were amplified. The spa gene was detected in 248 out of 250 isolates (99.2%), whereas in 2 isolates (0.8%), it remained undetected and referred as non-typable isolates. The 248 S. aureus isolates were typed into 39 spa types, which clustered into six different spa clonal clusters and eight singletons. CONCLUSION: High diversity observed within S. aureus isolates indicated that many different strains circulate in the study region or in the hospital. The results would contribute in the understanding of epidemiology related to S. aureus spread and prevention.

Otomycosis in a Rural Community Attending a Tertiary Care Hospital: Assessment of Risk Factors and Identification of Fungal and Bacterial Agents.

INTRODUCTION: Otomycosis is one of the frequently encountered fungal infections of external auditory canal, commonly seen in tropical and subtropical regions of the world. Various host (local, systemic) and environmental factors can predispose a person to otomycosis. Although clinical presentations along with otoscopic findings of the patients are well suggestive of fungal infection, proper identification of causative agents is mandatory in order to prevent recurrences and complications. AIM: To define the aetiology of clinically diagnosed otomycosis in rural population, to derive association of risk factors with otomycosis, to isolate and identify fungal and bacterial agents and to analyze the association between them. MATERIALS AND METHODS: A prospective study was conducted in the Department of Microbiology and Department of Ear, Nose and Throat (ENT), SGT hospital over eight months period. A total of 350 consecutive patients of more than five years of age with clinical diagnosis of otomycosis were included in the study. Demographic profile, predisposing factors, presenting complaints and clinical findings of clinically diagnosed patients were evaluated and analyzed. Samples were collected, transported and evaluated by both direct examination and culture method for bacteriological and mycological examination. RESULTS: Male to female ratio in study participants was 1.3:1. Mycological examination yielded 346 fungal isolates in 310 samples from a total of 350 clinically diagnosed cases of otomycosis. Self cleaning, instillation of mustard oil and use of ear drops appeared to be common predisposing factors in otomycosis. Significant association was observed between these practices and otomycosis. Aspergillus species was the predominant fungi followed by Candida spp., Penicillium, Mucor and Trichophyton mentagrophyte. 11% of the cases were found to have mixed fungal and bacterial infections. CONCLUSION: The present study highlights the highest isolation of Aspergillus complex in cases of clinically diagnosed otomycosis in a rural community with higher practice of self cleaning and using home remedies and eardrops to get relief from sensation of blocked ear and itching.

Simvastatin prevents and reverses depigmentation in a mouse model of vitiligo.

Vitiligo is a common autoimmune disease of the skin that results in disfiguring white spots. There are no Food and Drug Administration (FDA)-approved treatments, and current treatments are time-consuming, expensive, and of low efficacy. We sought to identify new treatments for vitiligo, and first considered repurposed medications because of the availability of safety data and expedited regulatory approval. We previously reported that the IFN-γ-induced chemokine CXCL10 is expressed in lesional skin from vitiligo patients, and that it is critical for the progression and maintenance of depigmentation in our mouse model of vitiligo. We hypothesized that targeting IFN-γ signaling might be an effective new treatment strategy. Activation of signal transducer and activator of transcription 1 (STAT1) is required for IFN-γ signaling and recent studies revealed that simvastatin, an FDA-approved cholesterol-lowering medication, inhibited STAT1 activation in vitro. Therefore, we hypothesized that simvastatin may be an effective treatment for vitiligo. We found that simvastatin both prevented and reversed depigmentation in our mouse model of vitiligo, and reduced the number of infiltrating autoreactive CD8(+) T cells in the skin. Treatment of melanocyte-specific, CD8(+) T cells in vitro decreased proliferation and IFN-γ production, suggesting additional effects of simvastatin directly on T cells. Based on these data, simvastatin may be a safe, targeted treatment option for patients with vitiligo.

CXCL10 is critical for the progression and maintenance of depigmentation in a mouse model of vitiligo.

Vitiligo is an autoimmune disease of the skin that results in disfiguring white spots. There are no U.S. Food and Drug Administration-approved treatments for vitiligo, and most off-label treatments yield unsatisfactory results. Vitiligo patients have increased numbers of autoreactive, melanocyte-specific CD8(+) T cells in the skin and blood, which are directly responsible for melanocyte destruction. We report that gene expression in lesional skin from vitiligo patients revealed an interferon-γ (IFN-γ)-specific signature, including the chemokine CXCL10. CXCL10 was elevated in both vitiligo patient skin and serum, and CXCR3, its receptor, was expressed on pathogenic T cells. To address the function of CXCL10 in vitiligo, we used a mouse model of disease that also exhibited an IFN-γ-specific gene signature, expression of CXCL10 in the skin, and up-regulation of CXCR3 on antigen-specific T cells. Mice that received Cxcr3(-/-) T cells developed minimal depigmentation, as did mice lacking Cxcl10 or treated with CXCL10-neutralizing antibody. CXCL9 promoted autoreactive T cell global recruitment to the skin but not effector function, whereas CXCL10 was required for effector function and localization within the skin. Surprisingly, CXCL10 neutralization in mice with established, widespread depigmentation induces reversal of disease, evidenced by repigmentation. These data identify a critical role for CXCL10 in both the progression and maintenance of vitiligo and thereby support inhibiting CXCL10 as a targeted treatment strategy.