Sensory deficit screen identifies nsf mutation that differentially affects SNARE recycling and quality control.

The AAA+ NSF complex is responsible for SNARE complex disassembly both before and after membrane fusion. Loss of NSF function results in pronounced developmental and degenerative defects. In a genetic screen for sensory deficits in zebrafish, we identified a mutation in nsf, I209N, that impairs hearing and balance in a dosage-dependent manner without accompanying defects in motility, myelination, and innervation. In vitro experiments demonstrate that while the I209N NSF protein recognizes SNARE complexes, the effects on disassembly are dependent upon the type of SNARE complex and I209N concentration. Higher levels of I209N protein produce a modest decrease in binary (syntaxin-SNAP-25) SNARE complex disassembly and residual ternary (syntaxin-1A-SNAP-25-synaptobrevin-2) disassembly, whereas at lower concentrations binary disassembly activity is strongly reduced and ternary disassembly activity is absent. Our study suggests that the differential effect on disassembly of SNARE complexes leads to selective effects on NSF-mediated membrane trafficking and auditory/vestibular function.

Diagnostic Value of Nucleocapsid Protein in Blood for SARS-CoV-2 Infection.

BACKGROUND: Biomarkers have been widely explored for coronavirus disease 2019 diagnosis. Both viral RNA or antigens (Ag) in the respiratory system and antibodies (Ab) in blood are used to identify active infection, transmission risk, and immune response but have limitations. This study investigated the diagnostic utility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (N-Ag) in serum. METHODS: We retrospectively studied 208 randomly selected cases with SARS-CoV-2 infection confirmed by viral RNA test in swabs. N-Ag concentrations were measured in remnant serum samples, compared to viral RNA or Ab results, and correlated to electronic health records for clinical value evaluation. RESULTS: Serum N-Ag was detected during active infection as early as day 2 from symptom onset with a diagnostic sensitivity of 81.5%. Within 1 week of symptom onset, the diagnostic sensitivity and specificity reached 90.9% (95% CI, 85.1%-94.6%) and 98.3% (95% CI, 91.1%-99.9%), respectively. Moreover, serum N-Ag concentration closely correlated to disease severity, reflected by highest level of care, medical interventions, chest imaging, and the length of hospital stays. Longitudinal analysis revealed the simultaneous increase of Abs and decline of N-Ag. CONCLUSIONS: Serum N-Ag is a biomarker for SARS-CoV-2 acute infection with high diagnostic sensitivity and specificity compared to viral RNA in the respiratory system. There is a correlation between serum N-Ag concentrations and disease severity and an inverse relationship of N-Ag and Abs. The diagnostic value of serum N-Ag, as well as technical and practical advantages it could offer, may meet unsatisfied diagnostic and prognostic needs during the pandemic.

Deep Neural Networks Offer Morphologic Classification and Diagnosis of Bacterial Vaginosis.

Bacterial vaginosis (BV) is caused by the excessive and imbalanced growth of bacteria in vagina, affecting 30 to 50% of women. Gram staining followed by Nugent scoring based on bacterial morphotypes under the microscope is considered the gold standard for BV diagnosis; this method is often labor-intensive and time-consuming, and results vary from person to person. We developed and optimized a convolutional neural network (CNN) model and evaluated its ability to automatically identify and classify three categories of Nugent scores from microscope images. The CNN model was first established with a panel of microscopic images with Nugent scores determined by experts. The model was trained by minimizing the cross-entropy loss function and optimized by using a momentum optimizer. The separate test sets of images collected from three hospitals were evaluated by the CNN model. The CNN model consisted of 25 convolutional layers, 2 pooling layers, and a fully connected layer. The model obtained 82.4% sensitivity and 96.6% specificity with the 5,815 validation images when altered vaginal flora and BV were considered the positive samples, which was better than the rates achieved by top-level technologists and obstetricians in China. The capability of our model for generalization was so strong that it exhibited 75.1% accuracy in three categories of Nugent scores on the independent test set of 1,082 images, which was 6.6% higher than the average of three technologists, who are hold bachelor's degrees in medicine and are qualified to make diagnostic decisions. When three technologists ran one specimen in triplicate, the precision of three categories of Nugent scores was 54.0%. One hundred three samples diagnosed by two technologists on different days showed a repeatability of 90.3%. The CNN model outperformed human health care practitioners in terms of accuracy and stability for three categories of Nugent score diagnosis. The deep learning model may offer translational applications in automating diagnosis of bacterial vaginosis with proper supporting hardware.

Novel DPY19L2 variants in globozoospermic patients and the overcoming this male infertility.

Globozoospermia has been reported to be a rare but severe causation of male infertility, which results from the failure of acrosome biogenesis and sperm head shaping. Variants of dpy-19-like 2 (DPY19L2) are highly related to globozoospermia, but related investigations have been mainly performed in patients from Western countries. Here, we performed a screening of DPY19L2 variants in a cohort of Chinese globozoospermic patients and found that five of nine patients carried DPY19L2 deletions and the other four patients contained novel DPY19L2 point mutations, as revealed by whole-exome sequencing. Patient 3 (P3) contained a heterozygous variant (c.2126+5G>A), P6 contained a homozygous nonsense mutation (c.1720C>T, p.Arg574*), P8 contained compound heterozygous variants (c.1182-1184delATC, p.Leu394_Ser395delinsPhe; c.368A>T, p.His123Arg), and P9 contained a heterozygous variant (c.1182-1184delATCTT, frameshift). We also reported intracytoplasmic sperm injection (ICSI) outcomes in the related patients, finding that ICSI followed by assisted oocyte activation (AOA) with calcium ionophore achieved high rates of live births. In summary, the infertility of these patients results from DPY19L2 dysfunction and can be treated by ICSI together with AOA.

Dopamine Modulates the Activity of Sensory Hair Cells.

The senses of hearing and balance are subject to modulation by efferent signaling, including the release of dopamine (DA). How DA influences the activity of the auditory and vestibular systems and its site of action are not well understood. Here we show that dopaminergic efferent fibers innervate the acousticolateralis epithelium of the zebrafish during development but do not directly form synapses with hair cells. However, a member of the D1-like receptor family, D1b, tightly localizes to ribbon synapses in inner ear and lateral-line hair cells. To assess modulation of hair-cell activity, we reversibly activated or inhibited D1-like receptors (D1Rs) in lateral-line hair cells. In extracellular recordings from hair cells, we observed that D1R agonist SKF-38393 increased microphonic potentials, whereas D1R antagonist SCH-23390 decreased microphonic potentials. Using ratiometric calcium imaging, we found that increased D1R activity resulted in larger calcium transients in hair cells. The increase of intracellular calcium requires Cav1.3a channels, as a Cav1 calcium channel antagonist, isradipine, blocked the increase in calcium transients elicited by the agonist SKF-38393. Collectively, our results suggest that DA is released in a paracrine fashion and acts at ribbon synapses, likely enhancing the activity of presynaptic Cav1.3a channels and thereby increasing neurotransmission. SIGNIFICANCE STATEMENT: The neurotransmitter dopamine acts in a paracrine fashion (diffusion over a short distance) in several tissues and bodily organs, influencing and regulating their activity. The cellular target and mechanism of the action of dopamine in mechanosensory organs, such as the inner ear and lateral-line organ, is not clearly understood. Here we demonstrate that dopamine receptors are present in sensory hair cells at synaptic sites that are required for signaling to the brain. When nearby neurons release dopamine, activation of the dopamine receptors increases the activity of these mechanosensitive cells. The mechanism of dopamine activation requires voltage-gated calcium channels that are also present at hair-cell synapses.

Tip-link protein protocadherin 15 interacts with transmembrane channel-like proteins TMC1 and TMC2.

The tip link protein protocadherin 15 (PCDH15) is a central component of the mechanotransduction complex in auditory and vestibular hair cells. PCDH15 is hypothesized to relay external forces to the mechanically gated channel located near its cytoplasmic C terminus. How PCDH15 is coupled to the transduction machinery is not clear. Using a membrane-based two-hybrid screen to identify proteins that bind to PCDH15, we detected an interaction between zebrafish Pcdh15a and an N-terminal fragment of transmembrane channel-like 2a (Tmc2a). Tmc2a is an ortholog of mammalian TMC2, which along with TMC1 has been implicated in mechanotransduction in mammalian hair cells. Using the above-mentioned two-hybrid assay, we found that zebrafish Tmc1 and Tmc2a can interact with the CD1 or CD3 cytoplasmic domain isoforms of Pcdh15a, and this interaction depends on the common region shared between the two Pcdh15 isoforms. Moreover, an interaction between mouse PCDH15-CD3 and TMC1 or TMC2 was observed in both yeast two-hybrid assays and coimmunoprecipitation experiments. To determine whether the Pcdh15-Tmc interaction is relevant to mechanotransduction in vivo, we overexpressed N-terminal fragments of Tmc2a in zebrafish hair cells. Overexpression of the Tmc2a N terminus results in mislocalization of Pcdh15a within hair bundles, together with a significant decrease in mechanosensitive responses, suggesting that a Pcdh15a-Tmc complex is critical for mechanotransduction. Together, these results identify an evolutionarily conserved association between the fish and mouse orthologs of PCDH15 and TMC1 and TMC2, supporting the notion that TMCs are key components of the transduction complex in hair cells.

Both pre- and postsynaptic activity of Nsf prevents degeneration of hair-cell synapses.

Vesicle fusion contributes to the maintenance of synapses in the nervous system by mediating synaptic transmission, release of neurotrophic factors, and trafficking of membrane receptors. N-ethylmaleimide-sensitive factor (NSF) is indispensible for dissociation of the SNARE-complex following vesicle fusion. Although NSF function has been characterized extensively in vitro, the in vivo role of NSF in vertebrate synaptogenesis is relatively unexplored. Zebrafish possess two nsf genes, nsf and nsfb. Here, we examine the function of either Nsf or Nsfb in the pre- and postsynaptic cells of the zebrafish lateral line organ and demonstrate that Nsf, but not Nsfb, is required for maintenance of afferent synapses in hair cells. In addition to peripheral defects in nsf mutants, neurodegeneration of glutamatergic synapses in the central nervous system also occurs in the absence of Nsf function. Expression of an nsf transgene in a null background indicates that stabilization of synapses requires Nsf function in both hair cells and afferent neurons. To identify potential targets of Nsf-mediated fusion, we examined the expression of genes implicated in stabilizing synapses and found that transcripts for multiple genes including brain-derived neurotrophic factor (bdnf) were significantly reduced in nsf mutants. With regard to trafficking of BDNF, we observed a striking accumulation of BDNF in the neurites of nsf mutant afferent neurons. In addition, injection of recombinant BDNF protein partially rescued the degeneration of afferent synapses in nsf mutants. These results establish a role for Nsf in the maintenance of synaptic contacts between hair cells and afferent neurons, mediated in part via the secretion of trophic signaling factors.

Ribeye is required for presynaptic Ca(V)1.3a channel localization and afferent innervation of sensory hair cells.

Ribbon synapses of the ear, eye and pineal gland contain a unique protein component: Ribeye. Ribeye consists of a novel aggregation domain spliced to the transcription factor CtBP2 and is one of the most abundant proteins in synaptic ribbon bodies. Although the importance of Ribeye for the function and physical integrity of ribbon synapses has been shown, a specific role in synaptogenesis has not been described. Here, we have modulated Ribeye expression in zebrafish hair cells and have examined the role of Ribeye in synapse development. Knockdown of ribeye resulted in fewer stimulus-evoked action potentials from afferent neurons and loss of presynaptic Ca(V)1.3a calcium channel clusters in hair cells. Additionally, afferent innervation of hair cells was reduced in ribeye morphants, and the reduction was correlated with depletion of Ribeye punctae. By contrast, transgenic overexpression of Ribeye resulted in Ca(V)1.3a channels colocalized with ectopic aggregates of Ribeye protein. Overexpression of Ribeye, however, was not sufficient to create ectopic synapses. These findings reveal two distinct functions of Ribeye in ribbon synapse formation--clustering Ca(V)1.3a channels at the presynapse and stabilizing contacts with afferent neurons--and suggest that Ribeye plays an organizing role in synaptogenesis.

Quantification of vestibular-induced eye movements in zebrafish larvae.

BACKGROUND: Vestibular reflexes coordinate movements or sensory input with changes in body or head position. Vestibular-evoked responses that involve the extraocular muscles include the vestibulo-ocular reflex (VOR), a compensatory eye movement to stabilize retinal images. Although an angular VOR attributable to semicircular canal stimulation was reported to be absent in free-swimming zebrafish larvae, recent studies reveal that vestibular-induced eye movements can be evoked in zebrafish larvae by both static tilts and dynamic rotations that tilt the head with respect to gravity. RESULTS: We have determined herein the basis of sensitivity of the larval eye movements with respect to vestibular stimulus, developmental stage, and sensory receptors of the inner ear. For our experiments, video recordings of larvae rotated sinusoidally at 0.25 Hz were analyzed to quantitate eye movements under infrared illumination. We observed a robust response that appeared as early as 72 hours post fertilization (hpf), which increased in amplitude over time. Unlike rotation about an earth horizontal axis, rotation about an earth vertical axis at 0.25 Hz did not evoke eye movements. Moreover, vestibular-induced responses were absent in mutant cdh23 larvae and larvae lacking anterior otoliths. CONCLUSIONS: Our results provide evidence for a functional vestibulo-oculomotor circuit in 72 hpf zebrafish larvae that relies upon sensory input from anterior/utricular otolith organs.

In vivo evidence for transdifferentiation of peripheral neurons.

It is commonly thought that differentiated neurons do not give rise to new cells, severely limiting the potential for regeneration and repair of the mature nervous system. However, we have identified cells in zebrafish larvae that first differentiate into dorsal root ganglia sensory neurons but later acquire a sympathetic neuron phenotype. These transdifferentiating neurons are present in wild-type zebrafish. However, they are increased in number in larvae that have a mutant voltage-gated sodium channel gene, scn8aa. Sodium channel knock-down promotes migration of differentiated sensory neurons away from the ganglia. Once in a new environment, sensory neurons transdifferentiate regardless of sodium channel expression. These findings reveal an unsuspected plasticity in differentiated neurons that points to new strategies for treatment of nervous system disease.

Synaptojanin1 is required for temporal fidelity of synaptic transmission in hair cells.

To faithfully encode mechanosensory information, auditory/vestibular hair cells utilize graded synaptic vesicle (SV) release at specialized ribbon synapses. The molecular basis of SV release and consequent recycling of membrane in hair cells has not been fully explored. Here, we report that comet, a gene identified in an ENU mutagenesis screen for zebrafish larvae with vestibular defects, encodes the lipid phosphatase Synaptojanin 1 (Synj1). Examination of mutant synj1 hair cells revealed basal blebbing near ribbons that was dependent on Cav1.3 calcium channel activity but not mechanotransduction. Synaptojanin has been previously implicated in SV recycling; therefore, we tested synaptic transmission at hair-cell synapses. Recordings of post-synaptic activity in synj1 mutants showed relatively normal spike rates when hair cells were mechanically stimulated for a short period of time at 20 Hz. In contrast, a sharp decline in the rate of firing occurred during prolonged stimulation at 20 Hz or stimulation at a higher frequency of 60 Hz. The decline in spike rate suggested that fewer vesicles were available for release. Consistent with this result, we observed that stimulated mutant hair cells had decreased numbers of tethered and reserve-pool vesicles in comparison to wild-type hair cells. Furthermore, stimulation at 60 Hz impaired phase locking of the postsynaptic activity to the mechanical stimulus. Following prolonged stimulation at 60 Hz, we also found that mutant synj1 hair cells displayed a striking delay in the recovery of spontaneous activity. Collectively, the data suggest that Synj1 is critical for retrieval of membrane in order to maintain the quantity, timing of fusion, and spontaneous release properties of SVs at hair-cell ribbon synapses.

Vesicular glutamate transporter 3 is required for synaptic transmission in zebrafish hair cells.

Hair cells detect sound and movement and transmit this information via specialized ribbon synapses. Here we report that asteroid, a gene identified in an ethylnitrosourea mutagenesis screen of zebrafish larvae for auditory/vestibular mutants, encodes vesicular glutamate transporter 3 (Vglut3). A splice site mutation in exon 2 of vglut3 results in a severe truncation of the predicted protein product and morpholinos directed against the vglut3 ATG start site or the affected splice junction replicate the asteroid phenotype. In situ hybridization shows that vglut3 is exclusively expressed in hair cells of the ear and lateral line organ. A second transporter gene, vglut1, is also expressed in zebrafish hair cells, but the level of vglut1 mRNA is not increased in the absence of Vglut3. Antibodies against Vglut3 label the basal end of hair cells and labeling is not present in asteroid/vglut3 mutants. Based on the localization of Vglut3 in hair cells, we suspected that the lack of vestibulo-ocular and acoustic startle reflexes in asteroid/vglut3 mutants was attributable to a defect in synaptic transmission in hair cells. In support of this notion, action currents in postsynaptic acousticolateralis neurons are absent in asteroid/vglut3 mutants. At the ultrastructural level, mutant asteroid/vglut3 hair cells show a decrease in the number of ribbon-associated synaptic vesicles, indicating a role for Vglut3 in synaptic vesicle biogenesis and/or tethering to the ribbon body. Lack of postsynaptic action currents in the mutants suggests that the remaining hair-cell synaptic vesicles contain insufficient levels of glutamate for generation of action potentials in first-order neurons.

Yeast AMID homologue Ndi1p displays respiration-restricted apoptotic activity and is involved in chronological aging.

Apoptosis-inducing factor (AIF) and AIF-homologous mitochondrion-associated inducer of death (AMID) are both mitochondrial flavoproteins that trigger caspase-independent apoptosis. Phylogenetic analysis suggests that these two proteins evolutionarily diverge back from their common prokaryote ancestor. Compared with AIF, the proapoptotic nature of AMID and its mode of action are much less clarified. Here, we show that overexpression of yeast AMID homologue internal NADH dehydrogenase (NDI1), but not external NADH dehydrogenase (NDE1), can cause apoptosis-like cell death, and this effect can be repressed by increased respiration on glucose-limited media. This result indicates that the regulatory network of energy metabolism, in particular the cross-talk between mitochondria and the rest of the cell, is involved in Ndi1p-induced yeast cell apoptosis. The apoptotic effect of NDI1 overexpression is associated with increased production of reactive oxygen species (ROS) in mitochondria. In addition, NDI1 overexpression in sod2 background causes cell lethality in both fermentable and semifermentable media. Interruption of certain components in the electron transport chain can suppress the growth inhibition from Ndi1p overexpression. We finally show that disruption of NDI1 or NDE1 decreases ROS production and elongates the chronological life span of yeast, accompanied by the loss of survival fitness. Implication of these findings for Ndi1p-induced apoptosis is discussed.

Yeast model uncovers dual roles of mitochondria in action of artemisinin.

Artemisinins, derived from the wormwood herb Artemisia annua, are the most potent antimalarial drugs currently available. Despite extensive research, the exact mode of action of artemisinins has not been established. Here we use yeast, Saccharamyces cerevisiae, to probe the core working mechanism of this class of antimalarial agents. We demonstrate that artemisinin's inhibitory effect is mediated by disrupting the normal function of mitochondria through depolarizing their membrane potential. Moreover, in a genetic study, we identify the electron transport chain as an important player in artemisinin's action: Deletion of NDE1 or NDI1, which encode mitochondrial NADH dehydrogenases, confers resistance to artemisinin, whereas overexpression of NDE1 or NDI1 dramatically increases sensitivity to artemisinin. Mutations or environmental conditions that affect electron transport also alter host's sensitivity to artemisinin. Sensitivity is partially restored when the Plasmodium falciparum NDI1 ortholog is expressed in yeast ndi1 strain. Finally, we showed that artemisinin's inhibitory effect is mediated by reactive oxygen species. Our results demonstrate that artemisinin's effect is primarily mediated through disruption of membrane potential by its interaction with the electron transport chain, resulting in dysfunctional mitochondria. We propose a dual role of mitochondria played during the action of artemisinin: the electron transport chain stimulates artemisinin's effect, most likely by activating it, and the mitochondria are subsequently damaged by the locally generated free radicals.