Collapse of late endosomal pH elicits a rapid Rab7 response via the V-ATPase and RILP.

Endosomal-lysosomal trafficking is accompanied by the acidification of endosomal compartments by the H+-V-ATPase to reach low lysosomal pH. Disruption of the correct pH impairs lysosomal function and the balance of protein synthesis and degradation (proteostasis). Here, we treated mammalian cells with the small dipeptide LLOMe, which is known to permeabilize lysosomal membranes, and find that LLOMe also impacts late endosomes (LEs) by neutralizing their pH without causing membrane permeabilization. We show that LLOMe leads to hyperactivation of Rab7 (herein referring to Rab7a), and disruption of tubulation and mannose-6-phosphate receptor (CI-M6PR; also known as IGF2R) recycling on pH-neutralized LEs. pH neutralization (NH4Cl) and expression of Rab7 hyperactive mutants alone can both phenocopy the alterations in tubulation and CI-M6PR trafficking. Mechanistically, pH neutralization increases the assembly of the V1G1 subunit (encoded by ATP6V1G1) of the V-ATPase on endosomal membranes, which stabilizes GTP-bound Rab7 via RILP, a known interactor of Rab7 and V1G1. We propose a novel pathway by which V-ATPase and RILP modulate LE pH and Rab7 activation in concert. This pathway might broadly contribute to pH control during physiologic endosomal maturation or starvation and during pathologic pH neutralization, which occurs via lysosomotropic compounds and in disease states.

Disruption of Golgi markers by two RILP-directed shRNAs in neurons: A new role for RILP or a neuron-specific off-target phenotype?

In neurons, degradation of dendritic cargos requires RAB7 and dynein-mediated retrograde transport to somatic lysosomes. To test if the dynein adapter RAB-interacting lysosomal protein (RILP) mediated the recruitment of dynein to late endosomes for retrograde transport in dendrites, we obtained several knockdown reagents previously validated in non-neuronal cells. Striking endosomal phenotypes elicited by one shRILP plasmid were not reproduced by another one. Furthermore, we discovered a profound depletion of Golgi/TGN markers for both shRILP plasmids. This Golgi disruption was only observed in neurons and could not be rescued by re-expression of RILP. This Golgi phenotype was also not found in neurons treated with siRILP or gRILP/Cas9. Lastly, we tested if a different RAB protein that interacts with RILP, namely the Golgi-associated RAB34, might be responsible for the loss of Golgi markers. Expression of a dominant-negative RAB34 did indeed cause changes in Golgi staining in a small subset of neurons but manifested as fragmentation rather than loss of staining. Unlike in non-neuronal cells, interference with RAB34 did not cause dispersal of lysosomes in neurons. Based on multiple lines of experimentation, we conclude that the neuronal Golgi phenotype observed with shRILP is likely off-target in this cell type specifically. Any observed disruptions of endosomal trafficking caused by shRILP in neurons might thus be downstream of Golgi disruption. It would be interesting to identify the actual target for this neuronal Golgi phenotype. Cell type-specific off-target phenotypes therefore likely occur in neurons, necessitating revalidation of reagents that were previously validated in other cell types.

Dynein Is Required for Rab7-Dependent Endosome Maturation, Retrograde Dendritic Transport, and Degradation.

In all cell types, endocytosed cargo is transported along a set of endosomal compartments, which are linked maturationally from early endosomes (EEs) via late endosomes (LEs) to lysosomes. Lysosomes are critical for degradation of proteins that enter through endocytic as well as autophagic pathways. Rab7 is the master regulator of early-to-late endosome maturation, motility, and fusion with lysosomes. We previously showed that most degradative lysosomes are localized in the soma and in the first 25 µm of the dendrite and that bulk degradation of dendritic membrane proteins occurs in/near the soma. Dendritic late endosomes therefore move retrogradely in a Rab7-dependent manner for fusion with somatic lysosomes. We now used cultured E18 rat hippocampal neurons of both sexes to determine which microtubule motor is responsible for degradative flux of late endosomes. Based on multiple approaches (inhibiting dynein/dynactin itself or inhibiting dynein recruitment to endosomes by expressing the C-terminus of the Rab7 effector, RILP), we now demonstrate that net retrograde flux of late endosomes in dendrites is supported by dynein. Inhibition of dynein also delays maturation of somatic endosomes, as evidenced by excessive accumulation of Rab7. In addition, degradation of dendritic cargos is inhibited. Our results also suggest that GDP-GTP cycling of Rab7 appears necessary not only for endosomal maturation but also for fusion with lysosomes subsequent to arrival in the soma. In conclusion, Rab7-dependent dynein/dynactin recruitment to dendritic endosomes plays multifaceted roles in dendritic endosome maturation as well as retrograde transport of late endosomes to sustain normal degradative flux.SIGNIFICANCE STATEMENT Lysosomes are critical for degradation of membrane and extracellular proteins that enter through endocytosis. Lysosomes are also the endpoint of autophagy and thus responsible for protein and organelle homeostasis. Endosomal-lysosomal dysfunction is linked to neurodegeneration and aging. We identify roles in dendrites for two proteins with links to human diseases, Rab7 and dynein. Our previous work identified a process that requires directional retrograde transport in dendrites, namely, efficient degradation of short-lived membrane proteins. Based on multiple approaches, we demonstrate that Rab7-dependent recruitment of dynein motors supports net retrograde transport to lysosomes and is needed for endosome maturation. Our data also suggest that GDP-GTP cycling of Rab7 is required for fusion with lysosomes and degradation, subsequent to arrival in the soma.

A dominant dendrite phenotype caused by the disease-associated G253D mutation in doublecortin (DCX) is not due to its endocytosis defect.

Doublecortin (DCX) is a protein needed for cortical development, and DCX mutations cause cortical malformations in humans. The microtubule-binding activity of DCX is well-described and is important for its function, such as supporting neuronal migration and dendrite growth during development. Previous work showed that microtubule binding is not sufficient for DCX-mediated promotion of dendrite growth and that domains in DCX's C terminus are also required. The more C-terminal regions of DCX bind several other proteins, including the adhesion receptor neurofascin and clathrin adaptors. We recently identified a role for DCX in endocytosis of neurofascin. The disease-associated DCX-G253D mutant protein is known to be deficient in binding neurofascin, and we now asked if disruption of neurofascin endocytosis underlies the DCX-G253D-associated pathology. We first demonstrated that DCX functions in endocytosis as a complex with both the clathrin adaptor AP-2 and neurofascin: disrupting either clathrin adaptor binding (DCX-ALPA) or neurofascin binding (DCX-G253D) decreased neurofascin endocytosis in primary neurons. We then investigated a known function for DCX, namely, increasing dendrite growth in cultured neurons. Surprisingly, we found that the DCX-ALPA and DCX-G253D mutants yield distinct dendrite phenotypes. Unlike DCX-ALPA, DCX-G253D caused a dominant-negative dendrite growth phenotype. The endocytosis defect of DCX-G253D thus was separable from its detrimental effects on dendrite growth. We recently identified Dcx-R59H as a dominant allele and can now classify Dcx-G253D as a second Dcx allele that acts dominantly to cause pathology, but does so via a different mechanism.

Different Doublecortin (DCX) Patient Alleles Show Distinct Phenotypes in Cultured Neurons: EVIDENCE FOR DIVERGENT LOSS-OF-FUNCTION AND "OFF-PATHWAY" CELLULAR MECHANISMS.

Doublecortin on the X-chromosome (DCX) is a neuronal microtubule-binding protein with a multitude of roles in neurodevelopment. In humans, DCX is a major genetic locus for X-linked lissencephaly. The best studied defects are in neuronal migration during corticogenesis and in the hippocampus, as well as axon and dendrite growth defects. Much effort has been directed at understanding the molecular and cellular bases of DCX-linked lissencephaly. The focus has been in particular on defects in microtubule assembly and bundling, using knock-out mice and expression of WT and mutant Dcx in non-neuronal cells. Dcx also binds other proteins besides microtubules, such as spinophilin (abbreviated spn; gene name Ppp1r9b protein phosphatase 1 regulatory subunit 9b) and the clathrin adaptors AP-1 and AP-2. Even though many non-sense and missense mutations of Dcx are known, their molecular and cellular defects are still only incompletely understood. It is also largely unknown how neurons are affected by expression of DCX patient alleles. We have now characterized several patient DCX alleles (DCX-R89G, DCX-R59H, DCX-246X, DCX-272X, and DCX-303X) using a gain-of-function dendrite growth assay in cultured rat neurons in combination with the determination of molecular binding activities and subcellular localization in non-neuronal and neuronal cells. First, we find that several mutants (Dcx-R89G and Dcx-272X) were loss-of-function alleles (as had been postulated) but surprisingly acted via different cellular mechanisms. Second, one allele (Dcx-R59H) formed cytoplasmic aggregates, which contained Hspa1B (heat shock protein 1B hsp70) and ubiquitinated proteins, trapped other cytoskeletal proteins, including spinophilin, and led to increased autophagy. This allele could thus be categorized as "off-pathway"/possibly neomorph. Our findings thus suggested that distinct DCX alleles caused dysfunction by different mechanisms.

Lessons learnt from 12 oral cholera vaccine campaigns in resource-poor settings.

Improving water and sanitation is the preferred choice for cholera control in the long-term. Nevertheless, vaccination is an available tool that has been shown to be a cost-effective option for cholera prevention in endemic countries or during outbreaks. In 2011 the first low-cost oral cholera vaccine for international use was given prequalification by the World Health Organization (WHO). To increase and prioritize use of the vaccine, WHO created a global stockpile in 2013 from which countries may request oral cholera vaccine for reactive campaigns. WHO has issued specific guidelines for applying for the vaccine, which was previously in short supply (despite prequalification for a second oral vaccine in 2015). The addition of a third WHO-prequalified oral cholera vaccine in 2016 is expected to increase the global stockpile considerably and alleviate supply issues. However, prioritization and best use of the vaccine (e.g. how, when and where to use) will remain challenges. We describe 12 past oral cholera vaccine campaigns, conducted in settings with varying burdens of cholera. These case studies illustrate three key challenges faced in the use of the oral cholera vaccines: regulatory hurdles, cold chain logistics and vaccine coverage and uptake. To pave the way for the introduction of current and future oral cholera vaccines, we discuss operational challenges and make recommendations for future research with respect to each of these challenges.

Améliorer l'accès à l'eau et à l'assainissement est le meilleur moyen de lutter contre le choléra à long terme. Néanmoins, la vaccination s'avère être un outil accessible et rentable pour la prévention du choléra dans les pays où cette maladie est endémique ou pendant des épidémies. En 2011, l'Organisation mondiale de la Santé (OMS) a présélectionné le premier vaccin anticholérique oral à faible coût destiné à un usage international. Afin de favoriser et de hiérarchiser l'usage de ce vaccin, l'OMS a créé en 2013 une réserve mondiale auprès de laquelle les pays peuvent demander des vaccins anticholériques oraux et mettre en œuvre des campagnes réactives. L'OMS a publié des directives spécifiques pour demander ce vaccin, qui n'était auparavant disponible qu'en quantité limitée (malgré la présélection d'un second vaccin oral en 2015). L'ajout, en 2016, d'un troisième vaccin anticholérique oral présélectionné par l'OMS devrait permettre d'augmenter sensiblement les réserves mondiales et d'atténuer les problèmes d'approvisionnement. Il restera cependant à traiter les questions de la hiérarchisation et du meilleur usage du vaccin (par ex., comment, à quel moment et à quel endroit l'utiliser). Nous décrivons ici 12 campagnes de vaccination orale contre le choléra qui ont été menées dans des régions diversement touchées par cette maladie. Ces études de cas illustrent trois grands défis qui se posent lors de l'utilisation de vaccins anticholériques oraux: les obstacles règlementaires, la logistique de la chaîne du froid et la couverture ainsi que le taux de vaccination. Afin de préparer l'introduction de vaccins anticholériques oraux, existants et futurs, nous examinons les difficultés opérationnelles et formulons des recommandations concernant de futurs travaux de recherche sur chacune de ces difficultés.

La mejora del agua y el saneamiento es la opción preferida para el control del cólera a largo plazo. Sin embargo, la vacunación es una herramienta disponible que ha demostrado ser una alternativa rentable para la prevención del cólera en países endémicos o durante brotes. En 2011, la Organización Mundial de la Salud (OMS) precalificó la primera vacuna anticolérica oral de bajo coste para uso internacional. Para aumentar y priorizar el uso de la vacuna, en 2013 la OMS creó una reserva global de la cual los países podían solicitar vacunas anticoléricas orales para campañas reactivas. La OMS ha publicado directrices específicas para la aplicación de la vacuna, cuyo suministro era escaso anteriormente (a pesar de la precalificación para una segunda vacuna oral en 2015). Está previsto que el hecho de añadir una tercera vacuna anticolérica oral precalificada por la OMS en 2016 aumente las reservas globales de forma considerable y reduzca los problemas de suministro. No obstante, la priorización y el buen uso de la vacuna (por ejemplo, cómo, cuándo y dónde utilizarla) seguirán siendo asuntos importantes. Se describen 12 campañas anteriores de vacunación oral contra el cólera, realizadas en entornos con distintos niveles de cólera. Estos estudios de casos ilustran los tres problemas principales que surgen al utilizar vacunas anticoléricas orales: obstáculos reglamentarios, logística de la gestión de la cadena de frío y cobertura y aceptación de la vacuna. Para allanar el terreno en la introducción de vacunas anticoléricas orales en el presente y en el futuro, se analizan las dificultades operativas y se presentan recomendaciones para futuras investigaciones con respecto a estos problemas.

Potential for Controlling Cholera Using a Ring Vaccination Strategy: Re-analysis of Data from a Cluster-Randomized Clinical Trial.

INTRODUCTION: Vaccinating a buffer of individuals around a case (ring vaccination) has the potential to target those who are at highest risk of infection, reducing the number of doses needed to control a disease. We explored the potential vaccine effectiveness (VE) of oral cholera vaccines (OCVs) for such a strategy. METHODS AND FINDINGS: This analysis uses existing data from a cluster-randomized clinical trial in which OCV or placebo was given to 71,900 participants in Kolkata, India, from 27 July to 10 September 2006. Cholera surveillance was then conducted on 144,106 individuals living in the study area, including trial participants, for 5 y following vaccination. First, we explored the risk of cholera among contacts of cholera patients, and, second, we measured VE among individuals living within 25 m of cholera cases between 8 and 28 d after onset of the index case. For the first analysis, individuals living around each index case identified during the 5-y period were assembled using a ring to define cohorts of individuals exposed to cholera index cases. An index control without cholera was randomly selected for each index case from the same population, matched by age group, and individuals living around each index control were assembled using a ring to define cohorts not exposed to cholera cases. Cholera attack rates among the exposed and non-exposed cohorts were compared using different distances from the index case/control to define the rings and different time frames to define the period at risk. For the VE analysis, the exposed cohorts were further stratified according to the level of vaccine coverage into high and low coverage strata. Overall VE was assessed by comparing the attack rates between high and low vaccine coverage strata irrespective of individuals' vaccination status, and indirect VE was assessed by comparing the attack rates among unvaccinated members between high and low vaccine coverage strata. Cholera risk among the cohort exposed to cholera cases was 5-11 times higher than that among the cohort not exposed to cholera cases. The risk gradually diminished with an increase in distance and time. The overall and indirect VE measured between 8 and 28 d after exposure to a cholera index case during the first 2 y was 91% (95% CI 62%-98%) and 93% (95% CI 44%-99%), respectively. VE persisted for 5 y after vaccination and was similar whether the index case was a young child (<5 y) or was older. Of note, this study was a reanalysis of a cholera vaccine trial that used two doses; thus, a limitation of the study relates to the assumption that a single dose, if administered quickly, will induce a similar level of total and indirect protection over the short term as did two doses. CONCLUSIONS: These findings suggest that high-level protection can be achieved if individuals living close to cholera cases are living in a high coverage ring. Since this was an observational study including participants who had received two doses of vaccine (or placebo) in the clinical trial, further studies are needed to determine whether a ring vaccination strategy, in which vaccine is given quickly to those living close to a case, is feasible and effective. TRIAL REGISTRATION: ClinicalTrials.gov NCT00289224.

Comparison of immune responses to a killed bivalent whole cell oral cholera vaccine between endemic and less endemic settings.

Studies on safety, immunogenicity and efficacy of the killed, bivalent whole cell oral cholera vaccine (Shanchol) have been conducted in historically endemic settings of Asia. Recent cholera vaccination campaigns in Haiti and Guinea have also demonstrated favourable immunogenicity and effectiveness in nonendemic outbreak settings. We performed a secondary analysis, comparing immune responses of Shanchol from two randomised controlled trials performed in an endemic and a less endemic area (Addis Ababa) during a nonoutbreak setting. While Shanchol may offer some degree of immediate protection in primed populations living in cholera endemic areas, as well as being highly immunogenic in less endemic settings, understanding the characteristics of immune responses in each of these areas is vital in determining ideal dosing strategies that offer the greatest public health impact to populations from areas with varying degrees of cholera endemicity.

Doublecortin (DCX) mediates endocytosis of neurofascin independently of microtubule binding.

Doublecortin on X chromosome (DCX) is one of two major genetic loci underlying human lissencephaly, a neurodevelopmental disorder with defects in neuronal migration and axon outgrowth. DCX is a microtubule-binding protein, and much work has focused on its microtubule-associated functions. DCX has other reported binding partners, including the cell adhesion molecule neurofascin, but the functional significance of the DCX-neurofascin interaction is not understood. Neurofascin localizes strongly to the axon initial segment in mature neurons, where it plays a role in assembling and maintaining other axon initial segment components. During development, neurofascin likely plays additional roles in axon guidance and in GABAergic synaptogenesis. We show here that DCX can modulate the surface distribution of neurofascin in developing cultured rat neurons and thereby the relative extent of accumulation between the axon initial segment and soma and dendrites. Mechanistically, DCX acts via increasing endocytosis of neurofascin from soma and dendrites. Surprisingly, DCX increases neurofascin endocytosis apparently independently of its microtubule-binding activity. We additionally show that the patient allele DCXG253D still binds microtubules but is deficient in promoting neurofascin endocytosis. We propose that DCX acts as an endocytic adaptor for neurofascin to fine-tune its surface distribution during neuronal development.

Oocyte specific oolemmal SAS1B involved in sperm binding through intra-acrosomal SLLP1 during fertilization.

Molecular mechanisms by which fertilization competent acrosome-reacted sperm bind to the oolemma remain uncharacterized. To identify oolemmal binding partner(s) for sperm acrosomal ligands, affinity panning was performed with mouse oocyte lysates using sperm acrosomal protein, SLLP1 as a target. An oocyte specific membrane metalloproteinase, SAS1B (Sperm Acrosomal SLLP1 Binding), was identified as a SLLP1 binding partner. cDNA cloning revealed six SAS1B splice variants, each containing a zinc binding active site and a putative transmembrane domain, with signal peptides in three variants. SAS1B transcripts were ovary specific. SAS1B protein was first detected in early secondary follicles in day 3 ovaries. Immunofluorescence localized SAS1B to the microvillar oolemma of M2 oocytes. After fertilization, SAS1B decreased on the oolemma and became virtually undetectable in blastocysts. In transfected CHO-K1 cells SAS1B localized to the surface of unpermeabilized cells. Recombinant and native SLLP1 co-localized with SAS1B to the microvillar domain of ovulated M2 oocytes. Molecular interactions between mouse SLLP1 and SAS1B were demonstrated by surface plasmon resonance, far-western, yeast two-hybrid, recombinant- and native-co-IP analyses. SAS1B bound to SLLP1 with high affinity. SAS1B had protease activity, and SAS1B protein or antibody significantly inhibited fertilization. SAS1B knockout female mice showed a 34% reduction in fertility. The study identified SAS1B-SLLP1 as a pair of novel sperm-egg binding partners involving the oolemma and intra-acrosomal compartment during fertilization.

"Disruption of Golgi markers by two RILP-directed shRNAs in neurons: a new role for RILP or a neuron-specific off-target phenotype?"

In neurons, degradation of dendritic cargos requires RAB7 and dynein-mediated retrograde transport to somatic lysosomes. In order to test if the dynein adaptor RILP (RAB-interacting lysosomal protein) mediated the recruitment of dynein to late endosomes for retrograde transport in dendrites, we obtained several knockdown reagents which had been previously validated in non-neuronal cells. We found that striking endosomal phenotypes elicited by one shRILP plasmid were not reproduced by another one. Furthermore, we discovered a profound depletion of Golgi/TGN markers for both shRILP plasmids. This Golgi disruption was only observed in neurons and could not be rescued by re-expression of RILP. This Golgi phenotype was also not found in neurons treated with siRILP or gRILP/Cas9. Lastly, we tested if a different RAB protein that interacts with RILP, namely the Golgi-associated RAB34, might be responsible for the loss of Golgi markers. Expression of a dominant-negative RAB34 did indeed cause changes in Golgi staining in a small subset of neurons but manifested as fragmentation rather than loss of markers. Unlike in non-neuronal cells, interference with RAB34 did not cause dispersal of lysosomes in neurons. Based on multiple lines of experimentation, we conclude that the neuronal Golgi phenotype observed with shRILP is likely off-target in this cell type specifically. Any observed disruptions of endosomal trafficking caused by shRILP in neurons might thus be downstream of Golgi disruption. Different approaches will be needed to test if RILP is required for late endosomal transport in dendrites. Cell type-specific off-target phenotypes therefore likely occur in neurons, making it prudent to re-validate reagents that were previously validated in other cell types.

Quantifying Single and Dual Channel Live Imaging Data: Kymograph Analysis of Organelle Motility in Neurons.

Live imaging is commonly used to study dynamic processes in cells. Many labs carrying out live imaging in neurons use kymographs as a tool. Kymographs display time-dependent microscope data (time-lapsed images) in two-dimensional representations showing position vs. time. Extraction of quantitative data from kymographs, often done manually, is time-consuming and not standardized across labs. We describe here our recent methodology for quantitatively analyzing single color kymographs. We discuss the challenges and solutions of reliably extracting quantifiable data from single-channel kymographs. When acquiring in two fluorescent channels, the challenge becomes analyzing two objects that may co-traffic together. One must carefully examine the kymographs from both channels and decide which tracks are the same or try to identify the coincident tracks from an overlay of the two channels. This process is laborious and time consuming. The difficulty in finding an available tool for such analysis has led us to create a program to do so, called KymoMerge. KymoMerge semi-automates the process of identifying co-located tracks in multi-channel kymographs and produces a co-localized output kymograph that can be analyzed further. We describe our analysis, caveats, and challenges of two-color imaging using KymoMerge.

Global loss of Neuron-specific gene 1 causes alterations in motor coordination, increased anxiety, and diurnal hyperactivity in male mice.

The Neuron-specific gene family (NSG1-3) consists of small endolysosomal proteins that are critical for trafficking multiple receptors and signaling molecules in neurons. NSG1 has been shown to play a critical role in AMPAR recycling from endosomes to plasma membrane during synaptic plasticity. However, to date nothing is known about whether NSG1 is required for normal behavior at an organismal level. Here we performed a battery of behavioral tests to determine whether loss of NSG1 would affect motor, cognitive, and/or affective behaviors, as well as circadian-related activity. Consistent with unique cerebellar expression of NSG1 among family members, we found that NSG1 was obligatory for motor coordination but not for gross motor function or learning. NSG1 knockout (KO) also altered performance across other behavioral modalities including anxiety-related and diurnal activity paradigms. Surprisingly, NSG1 KO did not cause significant impairments across all tasks within a given modality, but had specific effects within each modality. For instance, we found increases in anxiety-related behaviors in tasks with multiple stressors (e.g., elevation and exposure), but not those with a single main stressor (e.g., exposure). Interestingly, NSG1 KO animals displayed a significant increase in locomotor activity during subjective daytime, suggesting a possible impact on diurnal activity rhythms or vigilance. Surprisingly, loss of NSG1 had no effect on hippocampal-dependent learning despite previous studies showing deficits in CA1 long-term potentiation. Together, these findings do not support a role of NSG1 in hippocampal-dependent learning, but support a role in mediating proper neuronal function across amygdalar and cerebellar circuits.

A non-inferiority trial comparing two killed, whole cell, oral cholera vaccines (Cholvax vs. Shanchol) in Dhaka, Bangladesh.

Bangladesh remains cholera endemic with biannual seasonal peaks causing epidemics. At least 300,000 severe cases and over 4,500 deaths occur each year. The available oral cholera vaccineshave not yet been adopted for cholera control in Bangladesh due to insufficient number of doses available for endemic control. With a public private partnership, icddr,b initiated a collaboration between vaccine manufacturers in Bangladesh and abroad. A locally manufactured Oral Cholera Vaccine (OCV) named Cholvax became available for testing in Bangladesh. We evaluated the safety and immunogenicity of this locally produced Cholvax (Incepta Vaccine Ltd) inexpensive OCV comparatively to Shanchol (Shantha Biotechnics-Sanofi Pasteur) which is licensed in several countries. We conducted a randomized non-inferiority clinical trial of bivalent, killed oral whole-cell cholera vaccine Cholvax vs. Shanchol in the cholera-endemic area of Mirpur, Dhaka, among three different age cohorts (1-5, 6-17 and 18-45 years) between April 2016 and April 2017. Two vaccine doses were given at 14 days apart to 2,052 healthy participants. No vaccine-related serious adverse events were reported. There were no significant differences in the frequency of solicited (7.31% vs. 6.73%) and unsolicited (1.46% vs. 1.07%) adverse events reported between the Cholvax and Shanchol groups. Vibriocidal antibody responses among the overall population for O1 Ogawa (81% vs. 77%) and O1 Inaba (83% vs. 84%) serotypes showed that Cholvax was non-inferior to Shanchol, with the non-inferiority margin of -10%. For O1 Inaba, GMT was 462.60 (Test group), 450.84 (Comparator group) with GMR 1.02(95% CI: 0.92, 1.13). For O1 Ogawa, GMT was 419.64 (Test group), 387.22 (Comparator group) with GMR 1.12 (95% CI: 1.02, 1.23). Cholvax was safe and non-inferior to Shanchol in terms of immunogenicity in the different age groups. These results support public use of Cholvax to contribute for reduction of the cholera burden in Bangladesh. ClinicalTrials.gov number: NCT027425581.

Booster immunization with a fractional dose of Prevnar 13 affects cell-mediated immune response but not humoral immunity in CD-1 mice.

Achieving durable protective immunity following vaccination is dependent on many factors, including vaccine composition and antigen dose, and it has been investigated for various types of vaccines. Aim of the present study was to investigate the overall immune response elicited by two different booster doses in CD-1 mice, by exploiting the largely used 13-valent pneumococcal conjugate vaccine Prevnar 13® (PCV13). Immunization was performed by two primary doses of PCV13 two weeks apart, and a full or fractional (1/5) booster dose on week 10. Serotype-specific antibody titer, avidity, and opsonophagocytic activity were evaluated one week later, and compared to cell-mediated immunity (CMI) responses determined as the frequency of cytokines producing splenocytes by in vitro recall with the antigens (carrier protein and polysaccharides). Data showed that regardless of the booster dose, a comparable humoral response was produced, characterized by similar amounts of serotype-specific antibodies, with analog avidity and opsonophagocytic properties. On the other hand, when CMI was evaluated, the presence of CRM197-specific IL-5 and IL-2 producing cells was evident in splenocytes from mice immunized with the full dose, while in those immunized with the fractional booster dose, IFN-γ producing cells responsive to both protein and polysaccharide antigens were significantly increased, whereas the number of IL-5 and IL-2 positive cells remained unaffected. Overall the present findings show that PCV13 humoral response in mice is associated to a Th2 predominant response at the full booster dose, while the fractional one favors a mixed Th1/Th2 response, suggesting an important role of CMI besides measurement of functional protective antibodies, as an additional and important key information in vaccine development.

CABYR isoforms expressed in late steps of spermiogenesis bind with AKAPs and ropporin in mouse sperm fibrous sheath.

BACKGROUND: CABYR is a polymorphic calcium-binding protein of the sperm fibrous sheath (FS) which gene contains two coding regions (CR-A and CR-B) and is tyrosine as well as serine/threonine phosphorylated during in vitro sperm capacitation. Thus far, the detailed information on CABYR protein expression in mouse spermatogenesis is lacking. Moreover, because of the complexity of this polymorphic protein, there are no data on how CABYR isoforms associate and assemble into the FS. METHODS: The capacity of mouse CABYR isoforms to associate into dimers and oligomers, and the relationships between CABYR and other FS proteins were studied by gel electrophoresis, Western blotting, immunofluorescence, immunoprecipitation and yeast two-hybrid analyses. RESULTS: The predominant form of mouse CABYR in the FS is an 80 kDa variant that contains only CABYR-A encoded by coding region A. CABYR isoforms form dimers by combining the 80 kDa CABYR-A-only variant with the 50 kDa variant that contains both CABYR-A and CABYR-B encoded by full length or truncated coding region A and B. It is proposed that this step is followed by the formation of larger oligomers, which then participate in the formation of the supramolecular structure of the FS in mouse sperm. The initial expression of CABYR occurs in the cytoplasm of spermatids at step 11 of spermiogenesis and increases progressively during steps 12-15. CABYR protein gradually migrates into the sperm flagellum and localizes to the FS of the principal piece during steps 15-16. Deletion of the CABYR RII domain abolished the interaction between CABYR and AKAP3/AKAP4 but did not abolish the interaction between CABYR and ropporin suggesting that CABYR binds to AKAP3/AKAP4 by its RII domain but binds to ropporin through another as yet undefined region. CONCLUSIONS: CABYR expresses at the late stage of spermiogenesis and its isoforms oligomerize and bind with AKAPs and ropporin. These interactions strongly suggest that CABYR participates in the assembly of complexes in the FS, which may be related to calcium signaling.

Distribution of RNA binding protein MOEP19 in the oocyte cortex and early embryo indicates pre-patterning related to blastomere polarity and trophectoderm specification.

We report the cloning and characterization of MOEP19, a novel 19 kDa RNA binding protein that marks a defined cortical cytoplasmic domain in oocytes and provides evidence of mammalian oocyte polarity and a form of pre-patterning that persists in zygotes and early embryos through the morula stage. MOEP19 contains a eukaryotic type KH-domain, typical of the KH-domain type I superfamily of RNA binding proteins, and both recombinant and native MOEP19 bind polynucleotides. By immunofluorescence, MOEP19 protein was first detected in primary follicles throughout the ooplasm. As oocytes expanded in size during oogenesis, MOEP19 increased in concentration. MOEP19 localized in the ovulated egg and early zygote as a symmetrical spherical cortical domain underlying the oolemma, deep to the zone of cortical granules. MOEP19 remained restricted to a cortical cytoplasmic crescent in blastomeres of 2-, 4- and 8-cell embryos. The MOEP19 domain was absent in regions underlying cell contacts. In morulae, the MOEP19 domain was found at the apex of outer, polarized blastomeres but was undetectable in blastomeres of the inner cell mass. In early blastocysts, MOEP19 localized in both mural and polar trophectoderm and a subset of embryos showed inner cell mass localization. MOEP19 concentration dramatically declined in late blastocysts. When blastomeres of 4- to 8-cell stages were dissociated, the polarized MOEP19 domain assumed a symmetrically spherical localization, while overnight culture of dissociated blastomeres resulted in formation of re-aggregated embryos in which polarity of the MOEP19 domain was re-established at the blastomere apices. MOEP19 showed no evidence of translation in ovulated eggs, indicating that MOEP19 is a maternal effect gene. The persistence during early development of the MOEP19 cortical oocyte domain as a cortical crescent in blastomers suggests an intrinsic pre-patterning in the egg that is related to the apical-basolateral polarity of the embryo. Although the RNAs bound to MOEP19 are presently unknown, we predict that the MOEP19 domain directs RNAs essential for normal embryonic development to specific locations in the oocyte and early embryo.

Targeted deletion of Tssk1 and 2 causes male infertility due to haploinsufficiency.

Targeted deletion of Tssk1 and 2 resulted in male chimeras which produced sperm/spermatogenic cells bearing the mutant allele, however this allele was never transmitted to offspring, indicating infertility due to haploinsufficiency. Morphological defects in chimeras included failure to form elongated spermatids, apoptosis of spermatocytes and spermatids, and the appearance of numerous round cells in the epididymal lumen. Characterization of TSSK2 and its interactions with the substrate, TSKS, were further investigated in human and mouse. The presence of both kinase and substrate in the testis was confirmed, while persistence of both proteins in spermatozoa was revealed for the first time. In vivo binding interactions between TSSK2 and TSKS were established through co-immunoprecipitation of TSSK2/TSKS complexes from both human sperm and mouse testis extracts. A role for the human TSKS N-terminus in enzyme binding was defined by deletion mapping. TSKS immunoprecipitated from both mouse testis and human sperm extracts was actively phosphorylated. Ser281 was identified as a phosphorylation site in mouse TSKS. These results confirm both TSSK 2 and TSKS persist in sperm, define the critical role of TSKS' N-terminus in enzyme interaction, identify Ser 281 as a TSKS phosphorylation site and indicate an indispensable role for TSSK 1 and 2 in spermiogenesis.

TSKS concentrates in spermatid centrioles during flagellogenesis.

Centrosomal coiled-coil proteins paired with kinases play critical roles in centrosomal functions within somatic cells, however knowledge regarding gamete centriolar proteins is limited. In this study, the substrate of TSSK1 and 2, TSKS, was localized during spermiogenesis to the centrioles of post-meiotic spermatids, where it reached its greatest concentration during the period of flagellogenesis. This centriolar localization persisted in ejaculated human spermatozoa, while centriolar TSKS diminished in mouse sperm, where centrioles are known to undergo complete degeneration. In addition to the centriolar localization during flagellogenesis, mouse TSKS and the TSSK2 kinase localized in the tail and acrosomal regions of mouse epididymal sperm, while TSSK2 was found in the equatorial segment, neck and the midpiece of human spermatozoa. TSSK2/TSKS is the first kinase/substrate pair localized to the centrioles of spermatids and spermatozoa. Coupled with the infertility due to haploinsufficiency noted in chimeric mice with deletion of Tssk1 and 2 (companion paper) this centriolar kinase/substrate pair is predicted to play an indispensable role during spermiogenesis.

Biochemical and structural characterization of apolipoprotein A-I binding protein, a novel phosphoprotein with a potential role in sperm capacitation.

The physiological changes that sperm undergo in the female reproductive tract rendering them fertilization-competent constitute the phenomenon of capacitation. Cholesterol efflux from the sperm surface and protein kinase A (PKA)-dependent phosphorylation play major regulatory roles in capacitation, but the link between these two phenomena is unknown. We report that apolipoprotein A-I binding protein (AI-BP) is phosphorylated downstream to PKA activation, localizes to both sperm head and tail domains, and is released from the sperm into the media during in vitro capacitation. AI-BP interacts with apolipoprotein A-I, the component of high-density lipoprotein involved in cholesterol transport. The crystal structure demonstrates that the subunit of the AI-BP homodimer has a Rossmann-like fold. The protein surface has a large two compartment cavity lined with conserved residues. This cavity is likely to constitute an active site, suggesting that AI-BP functions as an enzyme. The presence of AI-BP in sperm, its phosphorylation by PKA, and its release during capacitation suggest that AI-BP plays an important role in capacitation possibly providing a link between protein phosphorylation and cholesterol efflux.