Clostridioides difficile phosphoproteomics shows an expansion of phosphorylated proteins in stationary growth phase

In this paper, we present a comprehensive analysis of protein phosphorylation in the Gram-positive enteropathogen Clostridioides difficile. To date, only limited evidence on the role of phosphorylation in the regulation of this organism has been published; the current study is expected to form the basis for research on this posttranslational modification in C. difficile.Phosphorylation is a posttranslational modification that can affect both housekeeping functions and virulence characteristics in bacterial pathogens. In the Gram-positive enteropathogen Clostridioides difficile, the extent and nature of phosphorylation events are poorly characterized, though a protein kinase mutant strain demonstrates pleiotropic phenotypes. Here, we used an immobilized metal affinity chromatography strategy to characterize serine, threonine, and tyrosine phosphorylation in C. difficile. We find limited protein phosphorylation in the exponential growth phase but a sharp increase in the number of phosphopeptides after the onset of the stationary growth phase. Our approach identifies expected targets and phosphorylation sites among the more than 1,500 phosphosites, including the protein kinase PrkC, the anti-sigma-F factor antagonist (SpoIIAA), the anti-sigma-B factor antagonist (RsbV), and HPr kinase/phosphorylase (HprK). Analysis of high-confidence phosphosites shows that phosphorylation on serine residues is most common, followed by threonine and tyrosine phosphorylation. This work forms the basis for a further investigation into the contributions of individual kinases to the overall phosphoproteome of C. difficile and the role of phosphorylation in C. difficile physiology and pathogenesis. IMPORTANCE In this paper, we present a comprehensive analysis of protein phosphorylation in the Gram-positive enteropathogen Clostridioides difficile. To date, only limited evidence on the role of phosphorylation in the regulation of this organism has been published; the current study is expected to form the basis for research on this posttranslational modification in C. difficile.Molecular basis of bacterial pathogenesis, virulence factors and antibiotic resistanc

Antibody-based inhibition of pathogenic new world hemorrhagic fever mammarenaviruses by steric occlusion of the human transferrin receptor 1 apical domain

Pathogenic clade B New World mammarenaviruses (NWM) can cause Argentine, Venezuelan, Brazilian, and Bolivian hemorrhagic fevers. Sequence variability among NWM glycoproteins (GP) poses a challenge to the development of broadly neutralizing therapeutics against the entire clade of viruses. However, blockade of their shared binding site on the apical domain of human transferrin receptor 1 (hTfR1/CD71) presents an opportunity for the development of effective and broadly neutralizing therapeutics. Here, we demonstrate that the murine monoclonal antibody OKT9, which targets the apical domain of hTfR1, can sterically block cellular entry by viral particles presenting clade B NWM glycoproteins (GP1-GP2). OKT9 blockade is also effective against viral particles pseudotyped with glycoproteins of a recently identified pathogenic Sabia-like virus. With nanomolar affinity for hTfR1, the OKT9 antigen binding fragment (OKT9-Fab) sterically blocks clade B NWM-GP1s and reduces infectivity of an attenuated strain of Junin virus. Binding of OKT9 to the hTfR1 ectodomain in its soluble, dimeric state produces stable assemblies that are observable by negative-stain electron microscopy. A model of the OKT9-sTfR1 complex, informed by the known crystallographic structure of sTfR1 and a newly determined structure of the OKT9 antigen binding fragment (Fab), suggests that OKT9 and the Machupo virus GP1 share a binding site on the hTfR1 apical domain. The structural basis for this interaction presents a framework for the design and development of high-affinity, broadly acting agents targeting clade B NWMs. IMPORTANCE Pathogenic clade B NWMs cause grave infectious diseases, the South American hemorrhagic fevers. Their etiological agents are Junin (JUNV), Guanarito (GTOV), Sabiá (SABV), Machupo (MACV), Chapare (CHAV), and a new Sabiá-like (SABV-L) virus recently identified in Brazil. These are priority A pathogens due to their high infectivity and mortality, their potential for person-to-person transmission, and the limited availability of effective therapeutics and vaccines to curb their effects. While low homology between surface glycoproteins of NWMs foils efforts to develop broadly neutralizing therapies targeting NWMs, this work provides structural evidence that OKT9, a monoclonal antibody targeting a single NWM glycoprotein binding site on hTfR1, can efficiently prevent their entry into cells.Fil: Ferrero, Sol. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Flores, Maria D.. University of California at Los Angeles; Estados UnidosFil: Short, Connor. University of California at Los Angeles; Estados UnidosFil: Vázquez, Cecilia Alejandra. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Clark, Lars E.. Harvard Medical School; Estados UnidosFil: Ziegenbein, James. University of California at Los Angeles; Estados UnidosFil: Zink, Samantha. University of California at Los Angeles; Estados UnidosFil: Fuentes, Daniel. University of California at Los Angeles; Estados UnidosFil: Payés, Cristian. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Batto, María V.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Collazo, Michael. University of California at Los Angeles; Estados UnidosFil: García, Cybele C.. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Abraham, Jonathan. Harvard Medical School; Estados Unidos. Brigham and Women's Hospital; Estados UnidosFil: Cordo, Sandra Myriam. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Rodriguez, Jose A.. University of California at Los Angeles; Estados UnidosFil: Helguera, Gustavo Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

Markov analysis of stochastic resonance in a periodically driven integrate-fire neuron

We model the dynamics of the leaky integrate-fire neuron under periodic stimulation as a Markov process with respect to the stimulus phase. This avoids the unrealistic assumption of a stimulus reset after each spike made in earlier work and thus solves the long-standing reset problem. The neuron exhibits stochastic resonance, both with respect to input noise intensity and stimulus frequency. The latter resonance arises by matching the stimulus frequency to the refractory time of the neuron. The Markov approach can be generalized to other periodically driven stochastic processes containing a reset mechanism.Comment: 23 pages, 10 figure

Triceps Surae Short Latency Stretch Reflexes Contribute to Ankle Stiffness Regulation during Human Running

During human running, short latency stretch reflexes (SLRs) are elicited in the triceps surae muscles, but the function of these responses is still a matter of controversy. As the SLR is primarily mediated by Ia afferent nerve fibres, various methods have been used to examine SLR function by selectively blocking the Ia pathway in seated, standing and walking paradigms, but stretch reflex function has not been examined in detail during running. The purpose of this study was to examine triceps surae SLR function at different running speeds using Achilles tendon vibration to modify SLR size. Ten healthy participants ran on an instrumented treadmill at speeds between 7 and 15 km/h under 2 Achilles tendon vibration conditions: no vibration and 90 Hz vibration. Surface EMG from the triceps surae and tibialis anterior muscles, and 3D lower limb kinematics and ground reaction forces were simultaneously collected. In response to vibration, the SLR was depressed in the triceps surae muscles at all speeds. This coincided with short-lasting yielding at the ankle joint at speeds between 7 and 12 km/h, suggesting that the SLR contributes to muscle stiffness regulation by minimising ankle yielding during the early contact phase of running. Furthermore, at the fastest speed of 15 km/h, the SLR was still depressed by vibration in all muscles but yielding was no longer evident. This finding suggests that the SLR has greater functional importance at slow to intermediate running speeds than at faster speeds

An Ensemble Analysis of Electromyographic Activity during Whole Body Pointing with the Use of Support Vector Machines

We explored the use of support vector machines (SVM) in order to analyze the ensemble activities of 24 postural and focal muscles recorded during a whole body pointing task. Because of the large number of variables involved in motor control studies, such multivariate methods have much to offer over the standard univariate techniques that are currently employed in the field to detect modifications. The SVM was used to uncover the principle differences underlying several variations of the task. Five variants of the task were used. An unconstrained reaching, two constrained at the focal level and two at the postural level. Using the electromyographic (EMG) data, the SVM proved capable of distinguishing all the unconstrained from the constrained conditions with a success of approximately 80% or above. In all cases, including those with focal constraints, the collective postural muscle EMGs were as good as or better than those from focal muscles for discriminating between conditions. This was unexpected especially in the case with focal constraints. In trying to rank the importance of particular features of the postural EMGs we found the maximum amplitude rather than the moment at which it occurred to be more discriminative. A classification using the muscles one at a time permitted us to identify some of the postural muscles that are significantly altered between conditions. In this case, the use of a multivariate method also permitted the use of the entire muscle EMG waveform rather than the difficult process of defining and extracting any particular variable. The best accuracy was obtained from muscles of the leg rather than from the trunk. By identifying the features that are important in discrimination, the use of the SVM permitted us to identify some of the features that are adapted when constraints are placed on a complex motor task

Functional Changes in Muscle Afferent Neurones in an Osteoarthritis Model: Implications for Impaired Proprioceptive Performance

Impaired proprioceptive performance is a significant clinical issue for many who suffer osteoarthritis (OA) and is a risk factor for falls and other liabilities. This study was designed to evaluate weight-bearing distribution in a rat model of OA and to determine whether changes also occur in muscle afferent neurones.Intracellular recordings were made in functionally identified dorsal root ganglion neurones in acute electrophysiological experiments on the anaesthetized animal following measurements of hind limb weight bearing in the incapacitance test. OA rats but not naïve control rats stood with less weight on the ipsilateral hind leg (P = 0.02). In the acute electrophysiological experiments that followed weight bearing measurements, action potentials (AP) elicited by electrical stimulation of the dorsal roots differed in OA rats, including longer AP duration (P = 0.006), slower rise time (P = 0.001) and slower maximum rising rate (P = 0.03). Depolarizing intracellular current injection elicited more APs in models than in naïve muscle afferent neurones (P = 0.01) indicating greater excitability. Axonal conduction velocity in model animals was slower (P = 0.04).The present study demonstrates changes in hind limb stance accompanied by changes in the functional properties of muscle afferent neurones in this derangement model of OA. This may provide a possible avenue to explore mechanisms underlying the impaired proprioceptive performance and perhaps other sensory disorders in people with OA

Genetic diagnosis of X-linked dominant hypophosphatemic rickets in a cohort study: Tubular reabsorption of phosphate and 1,25(OH)2D serum levels are associated with PHEX mutation type

<p>Abstract</p> <p>Background</p> <p>Genetic Hypophosphatemic Rickets (HR) is a group of diseases characterized by renal phosphate wasting with inappropriately low or normal 1,25-dihydroxyvitamin D₃(1,25(OH)₂D) serum levels. The most common form of HR is X-linked dominant HR (XLHR) which is caused by inactivating mutations in the <it>PHEX </it>gene. The purpose of this study was to perform genetic diagnosis in a cohort of patients with clinical diagnosis of HR, to perform genotype-phenotype correlations of those patients and to compare our data with other HR cohort studies.</p> <p>Methods</p> <p>Forty three affected individuals from 36 non related families were analyzed. For the genetic analysis, the <it>PHEX </it>gene was sequenced in all of the patients and in 13 cases the study was complemented by mRNA sequencing and Multiple Ligation Probe Assay. For the genotype-phenotype correlation study, the clinical and biochemical phenotype of the patients was compared with the type of mutation, which was grouped into clearly deleterious or likely causative, using the Mann-Whitney and Fisher's exact test.</p> <p>Results</p> <p>Mutations in the <it>PHEX </it>gene were identified in all the patients thus confirming an XLHR. Thirty four different mutations were found distributed throughout the gene with higher density at the 3' end. The majority of the mutations were novel (69.4%), most of them resulted in a truncated PHEX protein (83.3%) and were family specific (88.9%). Tubular reabsorption of phosphate (TRP) and 1,25(OH)₂D serum levels were significantly lower in patients carrying clearly deleterious mutations than in patients carrying likely causative ones (61.39 ± 19.76 vs. 80.14 ± 8.80%, p = 0.028 and 40.93 ± 30.73 vs. 78.46 ± 36.27 pg/ml, p = 0.013).</p> <p>Conclusions</p> <p><it>PHEX </it>gene mutations were found in all the HR cases analyzed, which was in contrast with other cohort studies. Patients with clearly deleterious <it>PHEX </it>mutations had lower TRP and 1,25(OH)₂D levels suggesting that the <it>PHEX </it>type of mutation might predict the XLHR phenotype severity.</p

What Is Stochastic Resonance? Definitions, Misconceptions, Debates, and Its Relevance to Biology

Stochastic resonance is said to be observed when increases in levels of unpredictable fluctuations—e.g., random noise—cause an increase in a metric of the quality of signal transmission or detection performance, rather than a decrease. This counterintuitive effect relies on system nonlinearities and on some parameter ranges being “suboptimal”. Stochastic resonance has been observed, quantified, and described in a plethora of physical and biological systems, including neurons. Being a topic of widespread multidisciplinary interest, the definition of stochastic resonance has evolved significantly over the last decade or so, leading to a number of debates, misunderstandings, and controversies. Perhaps the most important debate is whether the brain has evolved to utilize random noise in vivo, as part of the “neural code”. Surprisingly, this debate has been for the most part ignored by neuroscientists, despite much indirect evidence of a positive role for noise in the brain. We explore some of the reasons for this and argue why it would be more surprising if the brain did not exploit randomness provided by noise—via stochastic resonance or otherwise—than if it did. We also challenge neuroscientists and biologists, both computational and experimental, to embrace a very broad definition of stochastic resonance in terms of signal-processing “noise benefits”, and to devise experiments aimed at verifying that random variability can play a functional role in the brain, nervous system, or other areas of biology

Chemotherapy-induced hyaluronan production: a novel chemoresistance mechanism in ovarian cancer

Background: Hyaluronan (HA) an important component of the extracellular matrix, has been linked to tumor progression and drug resistance in several malignancies. However, limited data is available for ovarian cancer. This study investigated the role of hyaluronan (HA) and a potential link between the HA-CD44 pathway and membrane ATP binding cassette (ABC) transporter proteins in ovarian cancer chemoresistance. Methods: We investigated the ability of HA to block the cytotoxic effects of the chemotherapy drug carboplatin, and to regulate the expression of ABC transporters in ovarian cancer cells. We also examined HA serum levels in ovarian cancer patients prior to and following chemotherapy and assessed its prognostic relevance. Results: HA increased the survival of carboplatin treated ovarian cancer cells expressing the HA receptor, CD44 (OVCAR-5 and OV-90). Carboplatin significantly increased expression of HAS2, HAS3 and ABCC2 and HA secretion in ovarian cancer cell conditioned media. Serum HA levels were significantly increased in patients following platinum based chemotherapy and at both 1st and 2nd recurrence when compared with HA levels prior to treatment. High serum HA levels (>50 μg/ml) prior to chemotherapy treatment were associated with significantly reduced progression-free (P = 0.014) and overall survival (P = 0.036). HA production in ovarian cancer cells was increased in cancer tissues collected following chemotherapy treatment and at recurrence. Furthermore HA treatment significantly increased the expression of ABC drug transporters (ABCB3, ABCC1, ABCC2, and ABCC3), but only in ovarian cancer cells expressing CD44. The effects of HA and carboplatin on ABC transporter expression in ovarian cancer cells could be abrogated by HA oligomer treatment. Importantly, HA oligomers increased the sensitivity of chemoresistant SKOV3 cells to carboplatin. Conclusions: Our findings indicate that carboplatin chemotherapy induces HA production which can contribute to chemoresistance by regulating ABC transporter expression. The HA-CD44 signaling pathway is therefore a promising target in platinum resistant ovarian cancer.Carmela Ricciardelli, Miranda P Ween, Noor A Lokman, Izza A Tan, Carmen E Pyragius, and Martin K Oehle