Agonists of prostaglandin E2 receptors as potential first in class treatment for nephronophthisis and related ciliopathies.

Nephronophthisis (NPH) is an autosomal recessive tubulointerstitial nephropathy belonging to the ciliopathy disorders and known as the most common cause of hereditary end-stage renal disease in children. Yet, no curative treatment is available. The major gene, NPHP1, encodes a protein playing key functions at the primary cilium and cellular junctions. Using a medium-throughput drug-screen in NPHP1 knockdown cells, we identified 51 Food and Drug Administration-approved compounds by their ability to alleviate the cellular phenotypes associated with the loss of NPHP1; 11 compounds were further selected for their physicochemical properties. Among those compounds, prostaglandin E1 (PGE1) rescued ciliogenesis defects in immortalized patient NPHP1 urine-derived renal tubular cells, and improved ciliary and kidney phenotypes in our NPH zebrafish and Nphp1 knockout mouse models. Furthermore, Taprenepag, a nonprostanoid prostaglandin E2 receptor agonist, alleviated the severe retinopathy observed in Nphp1−/− mice. Finally, comparative transcriptomics allowed identification of key signaling pathways downstream PGE1, including cell cycle progression, extracellular matrix, adhesion, or actin cytoskeleton organization. In conclusion, using in vitro and in vivo models, we showed that prostaglandin E2 receptor agonists can ameliorate several of the pleotropic phenotypes caused by the absence of NPHP1; this opens their potential as a first therapeutic option for juvenile NPH-associated ciliopathies.

Ultrasonographic evaluation of the early brain growth pattern in very low birth weight infants.

BACKGROUND: Preterm infants develop smaller brain volumes compared to term newborns. Our aim is to study early brain growth related to perinatal factors in very low birth weight infants (VLBWI). METHODS: Manual segmentation of total brain volume (TBV) was performed in weekly 3D-ultrasonographies in our cohort of VLBWI. We studied the brain growth pattern related to term magnetic resonance image (term-MRI). RESULTS: We found different brain growth trajectories, with smaller brain volumes and a decrease in brain growth rate in those VLBWI who would later have an abnormal term-MRI (mean TBV 190.68 vs. 213.9 cm3; P = 0.0001 and mean TBV growth rate 14.35 (±1.27) vs. 16.94 (±2.29) cm3/week; P = 0.0001). TBV in those with normal term-MRI was related to gestational age (GA), being small for gestational age (SGA), sex, and duration of parenteral nutrition (TPN) while in those with abnormal term-MRI findings it was related to GA, SGA, TPN, and comorbidities. We found a deceleration in brain growth rate in those with ≥3 comorbidities. CONCLUSIONS: An altered brain growth pattern in VLBWI who subsequently present worst scores on term-MRI is related to GA, being SGA and comorbidities. Early ultrasonographic monitoring of TBV could be useful to detect deviated patterns of brain growth. IMPACT STATEMENT: We describe the brain growth pattern in very low birth weight infants during their first postnatal weeks. Brain growth may be affected in the presence of certain perinatal factors and comorbidities, conditioning a deviation of the normal growth pattern. The serial ultrasound follow-up of these at-risk patients allows identifying these brain growth patterns early, which offers a window of opportunity for implementing earlier interventions.

Relationship of early brain growth pattern measured by ultrasound with neurological outcome at two years of age in very low birth weight infants.

The purpose of this study is to define the impact of early brain growth trajectory in very low birth weight infants (VLBWI) on neurological prognosis at 2 years, assessed using sequential ultrasound (US) scans. This is a prospective cohort study with consecutive inclusion of VLBWI ≤ 32 weeks gestational age and ≤ 1500 g at birth. Total brain volume (TBV) was assessed using sequential 3D-US from birth to discharge. Prognosis at 2 years (corrected age) was assessed using the Bayley Scales of Infant and Toddler Development Third Edition. TBV showed slower growth with postmenstrual age (PMA) in those VLBWI who had an adverse cognitive prognosis compared to those with good cognitive prognosis (mean difference in TBV between prognostic groups from 4.56 cm3 at 28 weeks to 42.58 cm3 at 43 weeks) as well as in those with adverse language prognosis (mean difference in TBV from 2.21 cm3 at 28 weeks to 26.98 cm3 at 43 weeks) although other variables showed more impact than TBV on language prognosis (gestational age at birth, brain injury at term, and socioeconomic status). No association was found between TBV and motor prognosis. Brain growth rate was also significantly higher in those VLBWI who presented good cognitive scores (18.78 + (0.33 × (PMA-33)) cm3/week) compared to those with adverse cognitive outcome (13.73 + (0.64 × (PMA-33)) cm3/week).  Conclusion: Early altered brain growth is associated with poor cognitive prognosis at 2 years of age. Using sequential US monitoring, we can detect early brain growth deviation in patients who will have adverse cognitive outcomes. What is known: • The prediction of neurodevelopmental outcome of VLBWI is mostly based on the presence of brain injury in US and structural magnetic resonance imaging (MRI) at term. • Some studies have related brain volume measured on MRI at term with neurodevelopment outcome. What is new: • VLBWI with adverse cognitive prognosis at two years of age present smaller brain volumes detectable by sequential US during NICU admission. • Brain volume can be estimated from 2D and 3D US and has prognostic value in VLBWI.

Treatment Initiation, Substance Use Trajectories, and the Social Determinants of Health in Persons Living With HIV Seeking Medication for Opioid Use Disorder.

BACKGROUND: People living with HIV and opioid use disorder (OUD) are disproportionally affected by adverse socio-structural exposures negatively affecting health, which have shown inconsistent associations with uptake of medications for OUD (MOUD). This study aimed to determine whether social determinants of health (SDOH) were associated with MOUD uptake and trajectories of substance use in a clinical trial of people seeking treatment. METHODS: Data are from a 2018 to 2019 randomized trial comparing the effectiveness of different MOUD to achieve viral suppression among people living with HIV and OUD. SDOH were defined by variables mapping to Healthy People 2030 domains: education (Education Access and Quality), income (Economic Stability), homelessness (Neighborhood and Built Environment), criminal justice involvement (Social and Community Context), and recent SUD care (Health Care Access and Quality). Associations between SDOH and MOUD initiation were assessed with Cox proportional hazards models, and SDOH and substance use over time with generalized estimating equation models. RESULTS: Participants (N = 114) averaged 47 years old, 63% were male, 56% were Black, and 12% Hispanic. Participants reported an average of 2.3 out of 5 positive SDOH indicators (SD = 1.2). Stable housing was the most commonly reported SDOH (61%), followed by no recent criminal justice involvement (59%), having a high-school level education or greater (56%), income stability (45%), and recent SUD care (13%). Each additional favorable SDOH was associated with a 25% increase in the likelihood of MOUD initiation during the study period [adjusted HR = 1.25, 95% CI = (1.01, 1.55), P = .044]. Positive SDOH were also associated with a decrease in the odds of baseline opioid use and a greater reduction in opioid use during subsequent weeks of the study (P < .001 for a joint test of baseline and slope differences). CONCLUSIONS: Positive social determinants of health, in aggregate, may increase the likelihood of MOUD treatment initiation among people living with HIV and OUD.

Novel 2-aryl-4-aryloxyquinoline-based fungistatics for Mucor circinelloides. Biological evaluation of activity, QSAR and docking study.

Zygomycetes are ubiquitous saprophytes in natural environments which transform organic matter. Some zygomycetes of gender Mucor have attracted interest in health sector. Due to its ability as opportunistic microorganisms infecting immuno-compromised people and to the few available pharmacological treatments, the mucormycosis is receiving worldwide attention. Concerning to the pharmacological treatments, some triazole-based compounds such as fluconazole are extensively used. Nevertheless, we focused in the quinolines since they are broadly used models for the design and development of new synthetic antifungal agents. In this study, the fungistatic activity on M. circinelloides of various 2-aryl-4-aryloxyquinoline-based compounds was discovered, and in some cases, it resulted better than reference compound fluconazole. These quinoline derivatives were synthesized via the Csp2-O bond formation using diaryliodonium(III) salts chemistry. A QSAR study was carried out to quantitatively correlate the chemical structure of the tested compounds with their biological activity. Also, a docking study to identify a plausible action target of our more active quinolines was carried out. The results highlighted an increased activity with the fluorine- and nitro-containing derivatives. In light of the few mucormycosis pharmacological treatments, herein we present some non-described molecules with excellent in vitro activities and potential use in the mucormycosis treatment.

Lung ultrasound score has better diagnostic ability than NT-proBNP to predict moderate-severe bronchopulmonary dysplasia.

The N-terminal end of B-type natriuretic peptide (NT-proBNP) and lung ultrasound (LUS) score have been proven to be adequate early biomarkers of bronchopulmonary dysplasia (BPD) in preterm infants. Our aim was to study if the predictive capacity of each one is increased by analyzing them together. We included infants born before 32 weeks with NT-proBNP and LUS scores on the first day of life (DOL) and on the 3rd, 7th, and 14th DOL and compared the diagnostic ability for moderate-severe BPD (msBPD) of each biomarker and in combination. We also compared them with a multivariate model of msBPD using only clinical variables. The sample size was 133 patients, and twenty-seven (20%) developed msBPD. The LUS score on the 7th DOL had better performance than NT-proBNP at the same moment: area under the receiver operating characteristic curve (AUC) 0.83 (0.75-0.89) versus 0.66 (0.56-0.75), p = 0.003, without differences in the rest of the times studied. These values did not increase when using the combination of both. A multivariate regression model that included only clinical variables (birth weight and invasive mechanical ventilation (IMV) at the 7th DOL) predicted msBPD with the same AUC as after the addition of any of these biomarkers, neither together. CONCLUSION: The LUS score is a better predictor of msBPD on the 7th DOL than NT-proBNP in preterm infants born before 32 weeks, although they have similar diagnostic accuracy on the 1st, 3rd, and 14th DOL. Neither of them, nor together, have a better AUC for msBPD than a clinical model with birthweight and the need for IMV at the 7th DOL. WHAT IS KNOWN: • NT-proBNP and LUS score are early predictors of moderate-severe bronchopulmonary dysplasia (msBPD). WHAT IS NEW: • The combination of both NT-proBNP and LUS score does not increase the predictive ability of each separately.

[Guillain Barre syndrome associated with coronavac vaccine. Report of one case]. / Reporte de un caso de Síndrome de Guillain Barré post vacuna Coronavac: ¿rol causal o asociación temporal?

We report a 50-year-old woman with a history of celiac disease, who presented with lumbar pain and progressive flaccid tetraparesis 48 hours after the inoculation of the first dose of CoronaVac inactivated SARS-CoV-2 vaccine. CSF was normal and electrodiagnostic studies showed an axonal motor polyneuropathy. No other triggers were identified, and other etiologies were ruled out. The presentation was compatible with the AMAN (Acute Motor Axonal Neuropathy) subtype of GBS, and intravenous immunoglobulin halted the progression of symptoms. Intensive neurorehabilitation was performed. The patient was discharged five weeks after admission, walking with poles and climbing stairs with minimal assistance. To date no cases of inactivated SARSCoV-2 vaccine related GBS have been reported. Thus, description of its clinical presentation is relevant. We discuss the current evidence relating GBS with vaccines, highlighting that vaccine associated GBS is a controversial entity and causality must be interpreted cautiously given the actual COVID-19 pandemic context.

Saliva screening of health care workers for SARS-CoV-2 detection.

Health care workers (HCWs) are at high risk for SARS-CoV-2. In addition, pre-symptomatic or asymptomatic transmission accounts for around half of the cases. Saliva testing is an option to detect SARS-CoV-2 infection. To determine the performance of saliva samples for screening, HCWs were tested for SARS-CoV-2 by RT-PCR. Those with a positive result in saliva were tested by nasopharyngeal swabbing for viral RNA detection and blood collection to search for the presence of specific antibodies. In September-October 2020, 100 HCWs were enrolled and followed up. Six subjects (6%) tested positive in saliva. Of them, 5/6 were positive in a subsequent nasopharyngeal swab and 4/6 developed signs and symptoms compatible with COVID-19. Among the latter, 3 seroconverted while asymptomatic HCWs remained seronegative. Saliva screening was helpful for identifying SARS-CoV-2 infection in HCWs. This screening permitted rapid personnel isolation avoiding further transmission of the virus in the hospital setting.

Self-collected saliva for SARS-CoV-2 detection: A prospective study in the emergency room.

Current diagnostic standards involve severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in nasopharyngeal swabs (NPS), but saliva is an attractive and noninvasive option for diagnosis. The objectives were to determine the performance of saliva in comparison with NPS for detecting SARS-CoV-2 and to compare the optimized home brew reverse-transcription polymerase chain reaction (RT-PCR) with a commercial RT-PCR. Paired NPS and saliva specimens were prospectively collected and tested by RT-PCR from patients presenting at an emergency room with signs and symptoms compatible with coronavirus disease-2019. A total of 348 samples from 174 patients were tested by RT-PCR assays. Among 174 patients with symptoms, 63 (36%) were SARS-CoV-2 positive in NPS using the optimized home-brew PCR. Of these 63 patients, 61 (98%) were also positive in saliva. An additional positive SARS-CoV-2 saliva was detected in a patient with pneumonia. Kappa Cohen's coefficient agreement between NPS and saliva was 0.96 (95% confidence interval [CI], 0.90-0.99). Median Ct values in NPS versus saliva were 18.88 (interquartile range [IQR], 15.60-23.58; range, 11.97-38.10) versus 26.10 (IQR, 22.75-30.06; range, 13.78-39.22), respectively (p < .0001). The optimized home-brew RT-PCR demonstrated higher analytical and clinical sensitivity compared with the commercial RT-PCR assay. A high sensitivity (98%) and agreement (kappa 0.96) in saliva samples compared to NPS was demonstrated when using an optimized home-brew PCR even when the viral load in saliva was lower than in NPS. This noninvasive sample is easy to collect, requires less consumable and avoids discomfort to patients. Importantly, self-collection of saliva can diminish exposure to healthcare personnel.

Illinois Unidos: A Community Demands Equity, Justice, and Inclusion.

At the onset of the COVID-19 pandemic, neither government officials nor members of the news media fully grasped what was happening in the Latino community. Underreporting of COVID-19 cases led to a systematic neglect of the Latino population and resulted in disproportionately high rates of infection, hospitalization, and death. Illinois Unidos was formed to engage in community mobilization, health communication, advocacy, and policy work in response to inequalities exacerbated by COVID-19 in Latino communities in Illinois. (Am J Public Health. 2021;111(S3):S204-S207. https://doi.org/10.2105/AJPH.2021.306407).

Circulation of human coronaviruses OC43 and 229E in Córdoba, Argentina.

This is the first study of respiratory infections in Córdoba, Argentina, caused by endemic human coronavirus (HCoV)-OC43 and HCOV-229E, which circulated during 2011-2012 at a 3% rate, either as single or multiple infections. They were detected mainly in children, but HCoV-229E was also found in adults. HCoV-229E was detected in five out of 631 samples (0.8%), and HCoV-OC43 was found in 14 out of 631 (2.2%) samples. Clinical manifestations ranged from fever to respiratory distress, and a significant association of HCoV-229E with asthma was observed. Further studies and surveillance are needed to provide better clinical insights, early diagnosis, and medical care of patients, as well as to contribute to epidemiology modeling and prevention.

Evaluation of Plasmodium falciparum MSP10 and its development as a serological tool for the Peruvian Amazon region.

BACKGROUND: Different antigens are needed to characterize Plasmodium falciparum infection in terms of seroreactivity and targets for invasion inhibition, in order to guide and identify the proper use of such proteins as tools for the development of serological markers and/or as vaccine candidates. METHODS: IgG responses in 84 serum samples from individuals with P. falciparum infection [classified as symptomatic (Sym) or asymptomatic (Asym)], or acute Plasmodium vivax infection, from the Peruvian Amazon region, were evaluated by enzyme-linked immunosorbent assays specific for a baculovirus-produced recombinant protein P. falciparum Merozoite Surface Protein 10 (rMSP10) and for non-EGF region selected peptides of PfMSP10 selected by a bioinformatics tool (PfMSP10-1, PfMSP10-2 and PfMSP10-3). Monoclonal antibodies against the selected peptides were evaluated by western blotting, confocal microscopy and inhibition invasion assays. RESULTS: Seroreactivity analysis of the P. falciparum Sym- and Asym-infected individuals against rMSP10 showed a higher response as compared to the individuals with P. vivax acute infection. IgG responses against peptide PfMSP10-1 were weak. Interestingly high IgG response was found against peptide PfMSP10-2 and the combination of peptides PfMSP10-1 + PfMSP10-2. Monoclonal antibodies were capable of detecting native PfMSP10 on purified schizonts by western blot and confocal microscopy. A low percentage of inhibition of merozoite invasion of erythrocytes in vitro was observed when the monoclonal antibodies were compared with the control antibody against AMA-1 antigen. Further studies are needed to evaluate the role of PfMSP10 in the merozoite invasion. CONCLUSIONS: The rMSP10 and the PfMSP10-2 peptide synthesized for this study may be useful antigens for evaluation of P. falciparum malaria exposure in Sym and Asym individuals from the Peruvian Amazon region. Moreover, these antigens can be used for further investigation of the role of this protein in other malaria-endemic areas.

NTproBNP is a useful early biomarker of bronchopulmonary dysplasia in very low birth weight infants.

Bronchopulmonary dysplasia (BPD) is a severe complication of prematurity that impacts survival and neurodevelopment. Currently, no early marker exists which could help clinicians identify which preterm infants will develop BPD. Given the evidence that NTproBNP is elevated in children with BPD, we hypothesized that it could be used as an early marker of BPD development. We conducted a prospective cohort study including very low birth weight infants (VLBWI) admitted to our NICU between January 2015 and January 2017 in which we determined serial NTproBNP levels on days 1 and 3 and then weekly, until 49 days of life. A total of 101 patients were recruited (mean birth weight 1152 g (SD 247.5), mean gestational age 28.9 weeks (SD 1.9)). NTproBNP levels differed among infants who did and did not develop BPD from 14 to 35 days of life with the greatest difference on day 14 of life (non-BPD group (n = 86): 1155 (IQR 852-1908) pg/mL, BPD (n = 15): 9707 (IQR 3212-29,560) pg/mL; p = 0.0003). The presence of HsPDA did not account for higher levels of NTproBNP at day 14 (p = 0.165). We calculated an optimal cutoff point of 2264 pg/mL at 14 days of life (sensitivity 100%, specificity 86% and AUC 0.93).Conclusions: NTproBNP at 14 days of life could be used as an early marker of later BPD development in VLBWI. What is Known: • Children with BPD have elevated NTproBNP levels, which are related to the severity of BPD and the development of pulmonary hypertension. What is New: • NTproBNP at 14 days of life is higher in those who later develop BPD, regardless of the presence of hemodynamically significant patent ductus arteriosus. • A calculated cutoff point of 2264 pg/mL of NTproBNP at 14 days has a sensitivity of 100% and specificity of 86% in the prediction of BPD.

Reproducibility of the assessment of myocardial function through tissue Doppler imaging in very low birth weight infants.

OBJECTIVES: This study aims to analyze the variability between two trained neonatologists when performing consecutive echocardiograms using tissue Doppler imaging (TDI) and conventional methods in very low birth weight infant (VLBWI). METHODS: Two serial echocardiograms were performed in 30 VLBWI infants. The echocardiographic parameters analyzed were tricuspid annular plane systolic excursion (TAPSE), A', E', and S' waves, and myocardial performance index acquired by TDI (MPI-TDI) of both ventricles and shortening fraction (SF). The intra-observer and inter-observer agreements and the intra-operator agreement were analyzed using quantitative and qualitative statistical methods. RESULTS: The intra-observer agreement was very good, TAPSE, and TDI-derived parameters had an intra-class correlation (ICC) > 0.8. TDI-derived velocities had a coefficient of variation (COV) < 11%, while MPI-TDI had a COV between 20%-28%. The inter-observer agreement was excellent. There was greater variability when analyzing intra-operator agreement, with the least variable parameter being TAPSE. According to PABAK, the variability presented moderately substantial agreement. CONCLUSIONS: Tricuspid annular plane systolic excursion is very reproducible between observers and operators. Measurements of TDI wave velocities are more reproducible than MPI-TDI. TDI is sufficiently reproducible in the VLBWI if adequate training is performed, and guidelines are followed to obtain standardized echocardiographic images.

NT-proBNP as an Early Marker of Diastolic Ventricular Dysfunction in Very-Low-Birth-Weight Infants.

The objective is to examine the correlation between plasma levels of N-terminal pro-brain natriuretic peptide (NT-proBNP) and tissue Doppler imaging (TDI) echocardiographic parameters in the first 28 days of life in very-low-birth-weight infants (VLBWI). VLBWI admitted to the Neonatal Intensive Care Unit (NICU) at Hospital Puerta del Mar, Spain, from January 2015 to January 2017 were prospectively enrolled. Weekly determination of plasma NT-proBNP (pg/mL), and echocardiograms were done during the first 28 days of life. 101 preterm infants with a mean GA of 28.85 weeks (± 1.85 SD) and mean birth weight of 1152 g (± 247.4 SD) were included. A total of 483 echocardiograms and 139 NT-proBNP determinations were performed. We found a negative correlation between plasma NT-proBNP levels and diastolic velocities: mitral A' (ρ = - 0.15, p = 0.04), mitral E' (ρ = - 0.17, p = 0.02), tricuspid A' (ρ = - 0.20, p = 0.006), tricuspid E' (ρ = - 0.24, p = 0.0009). In the first 24 h of life, NT-proBNP levels were strongly correlated with mitral A' and E' velocities in patients with no patent ductus arteriosus (PDA) (ρ = - 0.75, p = 0.04). In preterm patients, elevated NT-proBNP levels are related to worse diastolic myocardial function. In the first 24 h, this correlation is much stronger in the absence of PDA.

CrpP Is a Novel Ciprofloxacin-Modifying Enzyme Encoded by the Pseudomonas aeruginosa pUM505 Plasmid.

The pUM505 plasmid, isolated from a clinical Pseudomonas aeruginosa isolate, confers resistance to ciprofloxacin (CIP) when transferred into the standard P. aeruginosa strain PAO1. CIP is an antibiotic of the quinolone family that is used to treat P. aeruginosa infections. In silico analysis, performed to identify CIP resistance genes, revealed that the 65-amino-acid product encoded by the orf131 gene in pUM505 displays 40% amino acid identity to the Mycobacterium smegmatis aminoglycoside phosphotransferase (an enzyme that phosphorylates and inactivates aminoglycoside antibiotics). We cloned orf131 (renamed crpP, for ciprofloxacin resistance protein, plasmid encoded) into the pUCP20 shuttle vector. The resulting recombinant plasmid, pUC-crpP, conferred resistance to CIP on Escherichia coli strain J53-3, suggesting that this gene encodes a protein involved in CIP resistance. Using coupled enzymatic analysis, we determined that the activity of CrpP on CIP is ATP dependent, while little activity against norfloxacin was detected, suggesting that CIP may undergo phosphorylation. Using a recombinant His-tagged CrpP protein and liquid chromatography-tandem mass spectrometry, we also showed that CIP was phosphorylated prior to its degradation. Thus, our findings demonstrate that CrpP, encoded on the pUM505 plasmid, represents a new mechanism of CIP resistance in P. aeruginosa, which involves phosphorylation of the antibiotic.

Nitrate assimilation pathway (NAP): role of structural (nit) and transporter (ntr1) genes in Fusarium oxysporum f.sp. lycopersici growth and pathogenicity.

The tomato pathogen Fusarium oxysporum f.sp. lycopersici possesses the capability to use nitrate as the only nitrogen source under aerobic and anaerobic conditions and to activate virulence-related functions when cultivated in the presence of nitrate, but not in ammonium. The genome of F. oxysporum f.sp. lycopersici encodes three paralogs nitrate reductase (NR) genes (nit1, nit2 and nit3) and one predicted ortholog of the Aspergillus nidulans high-affinity nitrate/nitrite transporters NtrA and NtrB, named ntr1. We set out to clarify the role of nit1, nit2, nit3 and ntr1 genes in nitrate assimilation and in the virulence of F. oxysporum f.sp. lycopersici. Quantitative RT-PCR analysis revealed that only nit1, nit2 and ntr1 are expressed at significant levels during growth in nitrate as the only nitrogen source. Targeted deletion of nit1 and ntr1, but not of nit2 or nit3, severely impaired growth of F. oxysporum on nitrate as nitrogen source, indicating that Nit1 and Ntr1 proteins are involved in nitrate assimilation by the fungus; biochemical analysis of nit mutants indicated that Nit1 and Nit2 enzymes contribute to about 50 and 30% of the total NR activity, respectively. In addition, a spontaneous chlorate-resistant mutant derived from F. oxysporum 4287, denoted NitFG, was characterized, showing inability to grow in nitrate under aerobic and anaerobic conditions and low levels of NR activity, in spite of its increased transcription levels of nit1 and nit2 genes. Tomato plant infection assays showed that NitFG and ∆ntr1 mutants induced an earlier death in tomato plants, whereas the single mutants ∆nit1, ∆nit2 and ∆nit1∆nit2 double mutant showed a mortality rate similar to the wild-type strain. Taken together, these results indicate that the Nit1 and Ntr1 proteins are important for nitrate assimilation of F. oxysporum f.sp. lycopersici incubated under aerobic and anaerobic conditions and that this metabolic process is not essential for the virulence of the fungus. These observations open new questions about the role of Nit1, Nit2, and Nit3 proteins in other routes of nitrate metabolism in this pathogenic fungus and in the possible regulatory role that can be exerted by the AreA protein in these routes.

Quality assurance for antimicrobial susceptibility testing of Neisseria gonorrhoeae in Latin American and Caribbean countries, 2013-2015.

OBJECTIVES: A Neisseria gonorrhoeae antimicrobial susceptibility quality control comparison programme was re-established in Latin America and the Caribbean to ensure antimicrobial susceptibility data produced from the region are comparable nationally and internationally. METHODS: Three panels, consisting of N. gonorrhoeae isolates comprising reference strains and other characterised isolates were sent to 11 participating laboratories between 2013 and 2015. Antimicrobial susceptibilities for these isolates were determined using agar dilution, Etest or disc diffusion methods. Modal minimum inhibitory concentrations (MICs) for each panel isolate/antibiotic combination were calculated. The guidelines of the Clinical and Laboratory Standards Institute were used for interpretations of antimicrobial susceptibility. The agreement of MICs with the modal MICs was determined for each of the participating laboratories as well as for each of the antibiotics tested. RESULTS: Five of 11 laboratories that participated in at least one panel had an overall average agreement between participants' MIC results and modal MICs of >90%. For other laboratories, agreements ranged from 60.0% to 82.4%. The proportion of agreement between interpretations for all the antibiotics, except penicillin and tetracycline, was >90%. The percentages of agreement between MIC results and their modes for erythromycin, spectinomycin, cefixime and azithromycin were >90%. Tetracycline, ceftriaxone and ciprofloxacin agreement ranged from 84.5% to 89.1%, while penicillin had 78.8% agreement between MICs and modal MICs. CONCLUSIONS: The participating laboratories had acceptable results, similar to other international quality assurance programmes. It is important to ensure continuation of the International Gonococcal Antimicrobial Susceptibility Quality Control Comparison Programme to ensure that participants can identify and correct any problems in antimicrobial susceptibility testing for N. gonorrhoeae as they arise and continue to generate reproducible and reliable data.

An aggregation-removal model for the formation and size determination of post-synaptic scaffold domains.

The formation and stability of synapses are key questions in neuroscience. Post-synaptic domains have been classically conceived as resulting from local insertion and turnover of proteins at the synapse. However, insertion is likely to occur outside the post-synaptic domains and advances in single-molecule imaging have shown that proteins diffuse in the plane of the membrane prior to their accumulation at synapses. We quantitatively investigated this scenario using computer simulations and mathematical analysis, taking for definiteness the specific case of inhibitory synapse components, i.e., the glycine receptor (GlyR) and the associated gephyrin scaffolding protein. The observed domain sizes of scaffold clusters can be explained by a dynamic balance between the aggregation of gephyrin proteins diffusing while bound to GlyR and their turnover at the neuron membrane. We also predict the existence of extrasynaptic clusters with a characteristic size distribution that significantly contribute to the size fluctuations of synaptic domains. New super-resolution data for gephyrin proteins established the existence of extrasynaptic clusters the sizes of which are consistent with the model predictions in a range of model parameters. At a general level, our results highlight aggregation with removal as a non-equilibrium phase separation which produces structures of tunable size.

Genetically encoding a light switch in an ionotropic glutamate receptor reveals subunit-specific interfaces.

Reprogramming receptors to artificially respond to light has strong potential for molecular studies and interrogation of biological functions. Here, we design a light-controlled ionotropic glutamate receptor by genetically encoding a photoreactive unnatural amino acid (UAA). The photo-cross-linker p-azido-L-phenylalanine (AzF) was encoded in NMDA receptors (NMDARs), a class of glutamate-gated ion channels that play key roles in neuronal development and plasticity. AzF incorporation in the obligatory GluN1 subunit at the GluN1/GluN2B N-terminal domain (NTD) upper lobe dimer interface leads to an irreversible allosteric inhibition of channel activity upon UV illumination. In contrast, when pairing the UAA-containing GluN1 subunit with the GluN2A subunit, light-dependent inactivation is completely absent. By combining electrophysiological and biochemical analyses, we identify subunit-specific structural determinants at the GluN1/GluN2 NTD dimer interfaces that critically dictate UV-controlled inactivation. Our work reveals that the two major NMDAR subtypes differ in their ectodomain-subunit interactions, in particular their electrostatic contacts, resulting in GluN1 NTD coupling more tightly to the GluN2B NTD than to the GluN2A NTD. It also paves the way for engineering light-sensitive ligand-gated ion channels with subtype specificity through the genetic code expansion.