Enteropathogenic Escherichia coli remodels host endosomes to promote endocytic turnover and breakdown of surface polarity.

Enteropathogenic E. coli (EPEC) is an extracellular diarrheagenic human pathogen which infects the apical plasma membrane of the small intestinal enterocytes. EPEC utilizes a type III secretion system to translocate bacterial effector proteins into its epithelial hosts. This activity, which subverts numerous signaling and membrane trafficking pathways in the infected cells, is thought to contribute to pathogen virulence. The molecular and cellular mechanisms underlying these events are not well understood. We investigated the mode by which EPEC effectors hijack endosomes to modulate endocytosis, recycling and transcytosis in epithelial host cells. To this end, we developed a flow cytometry-based assay and imaging techniques to track endosomal dynamics and membrane cargo trafficking in the infected cells. We show that type-III secreted components prompt the recruitment of clathrin (clathrin and AP2), early (Rab5a and EEA1) and recycling (Rab4a, Rab11a, Rab11b, FIP2, Myo5b) endocytic machineries to peripheral plasma membrane infection sites. Protein cargoes, e.g. transferrin receptors, ß1 integrins and aquaporins, which exploit the endocytic pathways mediated by these machineries, were also found to be recruited to these sites. Moreover, the endosomes and cargo recruitment to infection sites correlated with an increase in cargo endocytic turnover (i.e. endocytosis and recycling) and transcytosis to the infected plasma membrane. The hijacking of endosomes and associated endocytic activities depended on the translocated EspF and Map effectors in non-polarized epithelial cells, and mostly on EspF in polarized epithelial cells. These data suggest a model whereby EPEC effectors hijack endosomal recycling mechanisms to mislocalize and concentrate host plasma membrane proteins in endosomes and in the apically infected plasma membrane. We hypothesize that these activities contribute to bacterial colonization and virulence.

Human Rabies - Utah, 2018.

On November 3, 2018, the Utah Department of Health (UDOH) was notified of a suspected human rabies case in a man aged 55 years. The patient's symptoms had begun 18 days earlier, and he was hospitalized for 15 days before rabies was suspected. As his symptoms worsened, he received supportive care, but he died on November 4. On November 7, a diagnosis of rabies was confirmed by CDC. This was the first documented rabies death in a Utah resident since 1944. This report summarizes the patient's clinical course and the subsequent public health investigation, which determined that the patient had handled several bats in the weeks preceding symptom onset. Public health agencies, in partnership with affected health care facilities, identified and assessed the risk to potentially exposed persons, facilitated receipt of postexposure prophylaxis (PEP), and provided education to health care providers and the community about the risk for rabies associated with bats. Human rabies is rare and almost always fatal. The findings from this investigation highlight the importance of early recognition of rabies, improved public awareness of rabies in bats, and the use of innovative tools after mass rabies exposure events to ensure rapid and recommended risk assessment and provision of PEP.

A novel solution for freezing small numbers of spermatozoa using a sperm vitrification device.

STUDY QUESTION: Does a novel sperm vitrification device (SpermVD) provide an efficient method for freezing a small number of human spermatozoa from men suffering from non-obstructive azoospermia? SUMMARY ANSWER: The novel SpermVD is an efficient and simple carrier method for freezing a small number of spermatozoa in low-volume droplets, reducing post-thaw search time from hours to minutes, allowing a 96% recovery rate and leading to successful use of sperm for fertilization. WHAT IS KNOWN ALREADY: Previous methods for cryopreservation of small numbers of human spermatozoa (e.g. mini-straws, ICSI pipette, alginate beads, cryoloop) have been proposed as a solution for cases of severe male infertility. Many drawbacks have prevented their widespread use, including cumbersome preparation and sperm retrieval procedures, and the fact that the thawed spermatozoa are not immediately available for micromanipulation and required additional treatment which posed excess risk of harm. STUDY DESIGN, SIZE, DURATION: We conducted a feasibility experiment of the novel SpermVD and a prospective cohort study of ICSI cycles in men suffering from non-obstructive azoospermia in two outpatient fertility IVF clinics, from 2015 through 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS: All patients underwent extended ejaculate search prior to the day of oocyte retrieval, and any single motile spermatozoa found was transferred to 0.8 µl droplets of 1:1 washing medium/cryoprotectant on the SpermVD, then plunged into liquid nitrogen for cryopreservation. In patients with non-obstructive azoospermia who underwent surgical TESE, both the motile and immotile spermatozoa found underwent cryopreservation using the SpermVD. On the day of oocyte retrieval, the SpermVD was thawed, directly transfered to the ICSI plate and retrieved spermatozoa were used for the ICSI procedure. MAIN RESULTS AND THE ROLE OF CHANCE: The prospective cohort included 44 cases. We used the SpermVD to vitrify 631 spermatozoa, of which 540 (86%) were motile. The average number of frozen spermatozoa per patient was 14.3 ± 9.3. After thawing, we retrieved 607 spermatozoa, producing a recovery rate of 96%. The average number of thawed spermatozoa was 13.8 ± 9.2. The recovery of 180 thawed motile sperm accounted for 33% of all frozen motile spermatozoa. The fertilization rate was 59%. Of 44 oocyte retrieval procedures, 24 (55%) clinical pregnancies were achieved. The delivery rate (not including three ongoing pregnancies) was 32% and the miscarriage rate was 29%. LIMITATIONS, REASONS FOR CAUTION: Although we presented the SpermVD on 44 cases, a larger cohort would provide more information. Moreover, we cryopreserved only motile sperm from the ejaculates and not immotile sperm, thus limiting the knowledge regarding the efficacy of the VD for immotile sperm from this source. WIDER IMPLICATIONS OF THE FINDINGS: The novel SpermVD is a simple efficient carrier, optimizing the protocol for freezing a small number of spermatozoa. It may allow for the routine use of frozen spermatozoa after TESE for men suffering from non-obstructive azoospermia and thus avoid repeated TESE surgeries. Furthermore, in cases of non-obstructive azoospermia, routine cryopreservation of the retrieved spermatozoa prior to the IVF cycle may avoid the risk of cycle cancelation and thus decrease the number of unnecessary oocyte retrieval procedures. STUDY FUNDING/COMPETING INTEREST(S): There was no external funding. There are no competing interests. TRIAL REGISTRATION NUMBER: IRB no 00119-16-ASMC.

Role and regulation of Glycogen synthase kinase-3 beta in bovine spermatozoa.

The serine/threonine kinase Glycogen synthase kinase 3 (GSK-3) is a master switch that regulates a multitude of cellular pathways, including the acrosome reaction in sperm. In epididymal sperm cells, for example, GSK-3 activity correlates with inhibition of motility-yet no direct pathways connecting GSK-3 activation with loss of motility have been described. Indeed, the details of how GSK-3 is regulated during sperm capacitation and the acrosome reaction remains obscure. To this end, we addressed the involvement of the GSK-3 beta isoform in several known pathways that contribute to motility and the acrosome reaction. We established that Protein kinase A (PKA) is the main regulator of GSK-3ß in sperm, as pre-treatment of cells with a GSK-3 inhibitor prior to addition of H89, an inhibitor of PKA, attenuated the motility loss induced by blocking PKA activity. Both induced and spontaneous acrosome reactions also occurred less frequently in sperm treated with GSK-3 inhibitors. Finally, we observed a slow decline in phosphorylation of GSK-3ß on Ser 9, which represents an inhibited state, during sperm capacitation; this phenotype is reversed during the induced acrosome reaction, in parallel to activation of Protein phosphatase 1. These results suggest that maintenance of sperm motility and acrosome reaction timing are mediated by PKA through the regulation of GSK-3 beta activity. Mol. Reprod. Dev. 84: 8-18, 2017. © 2016 Wiley Periodicals, Inc.

Evidence of autoinflammation as a principal mechanism of myocardial injury in SARS-CoV-2 PCR-positive medical examiner cases.

BACKGROUND: Disease from Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) remains the seventh leading cause of death in the United States. Many patients infected with this virus develop later cardiovascular complications including myocardial infarctions, stroke, arrhythmia, heart failure, and sudden cardiac death (20-28%). The purpose of this study is to understand the primary mechanism of myocardial injury in patients infected with SARS-CoV-2. METHODS: We investigated a consecutive cohort of 48 medical examiner cases who died with PCR-positive SARS-CoV-2 (COVpos) infection in 2020. We compared them to a consecutive cohort of 46 age- and sex-matched controls who were PCR-negative for SARS-CoV-2 (COVneg). Clinical information available at postmortem examination was reviewed on each patient. Formalin-fixed sections were examined using antibodies directed against CD42 (platelets), CD15 (myeloid cells), CD68 (monocytes), C4d, fibrin, CD34 (stem cell antigen), CD56 (natural killer cells), and myeloperoxidase (MPO) (neutrophils and neutrophil extracellular traps(NETs)). We used a Welch 2-sample T-test to determine significance. A cluster analysis of marker distribution was also done. RESULTS: We found a significant difference between COVpos and COVneg samples for CD42, CD15, CD68, C4d, fibrin, and MPO, all of which were significant at p < 0.001. The most prominent features were neutrophils (CD15, MPO) and MPO-positive debris suggestive of NETs. A similar distribution of platelets, monocytes, fibrin and C4d was seen in COVpos cases. Clinical features were similar in COVpos and COVneg cases for age, sex, and body mass index (BMI). CONCLUSION: These findings suggest an autoinflammatory process is likely involved in cardiac damage during SARS-CoV-2 infection. No information about clinical cardiac disease was available.

Neurotransmission and neuromodulation systems in the learning and memory network of Octopus vulgaris.

The vertical lobe (VL) in the octopus brain plays an essential role in its sophisticated learning and memory. Early anatomical studies suggested that the VL is organized in a "fan-out fan-in" connectivity matrix comprising only three morphologically identified neuron types; input axons from the median superior frontal lobe (MSFL) innervating en passant millions of small amacrine interneurons (AMs), which converge sharply onto large VL output neurons (LNs). Recent physiological studies confirmed the feedforward excitatory connectivity; a glutamatergic synapse at the first MSFL-to-AM synaptic layer and a cholinergic AM-to-LNs synapse. MSFL-to-AMs synapses show a robust hippocampal-like activity-dependent long-term potentiation (LTP) of transmitter release. 5-HT, octopamine, dopamine and nitric oxide modulate short- and long-term VL synaptic plasticity. Here, we present a comprehensive histolabeling study to better characterize the neural elements in the VL. We generally confirmed glutamatergic MSFLs and cholinergic AMs. Intense labeling for NOS activity in the AMs neurites were in-line with the NO-dependent presynaptic LTP mechanism at the MSFL-to-AM synapse. New discoveries here reveal more heterogeneity of the VL neurons than previously thought. GABAergic AMs suggest a subpopulation of inhibitory interneurons in the first input layer. Clear γ-amino butyric acid labeling in the cell bodies of LNs supported an inhibitory VL output, yet the LNs co-expressed FMRFamide-like neuropeptides, suggesting an additional neuromodulatory role of the VL output. Furthermore, a group of LNs was glutamatergic. A new cluster of cells organized as a "deep nucleus" showed rich catecholaminergic labeling and may play a role in intrinsic neuromodulation. In-situ hybridization and immunolabeling allowed characterization and localization of a rich array of neuropeptides and neuromodulators, likely involved in reward/punishment signals. This analysis of the fast transmission system, together with the newly found cellular elements, help integrate behavioral, physiological, pharmacological and connectome findings into a more comprehensive understanding of an efficient learning and memory network.

The possibility of integrating motile sperm organelle morphology examination (MSOME) with intracytoplasmic morphologically-selected sperm injection (IMSI) when treating couples with unexplained infertility.

PURPOSE: To examine the efficacy of motile sperm organelle morphology examination (MSOME) and intracytoplasmic morphologically-selected sperm injection (IMSI) for unexplained infertility. METHODS: This historical study, included 271 couples with primary, unexplained infertility/male subfertility, treated at an outpatient, IVF clinic, 2015-2018. These couples underwent MSOME after ≥3 failed intrauterine insemination (IUI) cycles and ≥1 failed IVF-ICSI cycle. They proceeded to intracytoplasmic morphologically-selected sperm injection (IMSI) within 6 months of MSOME. IMSI is conducted on the day of oocyte pick-up with a fresh semen sample. Pregnancy and delivery rates were analyzed. RESULTS: The cohort was divided based on percentage of normal cells at MSOME: Group A included 55 with no normal cells, Group B, 184 with 0.5%≤ normal cells ≤1.5% and Group C, 32 with ≥2% normal cells. Normal spermatozoa were found in 49 (89%) of Group A after extensive search. Group A had higher pregnancy rate (62.7%) compared to B (47.2%, P = 0.05) and C (28.1%, P = 0.002). Group B had higher pregnancy rate than C (p = 0.045). Delivery rate was higher in Group A (52.1%) compared to B (34.1%, p = 0.023) and C (21.9%, p = 0.007). Pregnancy and delivery rates were higher in A compared to B+C (p = 0.018, p = 0.01, respectively). CONCLUSIONS: MSOME may be useful for evaluating unexplained infertility. IMSI can be recommended for men with <2% normal spermatozoa at MSOME.

Optimizing the protocol for vitrification of individual spermatozoa by adjusting equilibration time.

Efficient cryopreservation of small numbers of human spermatozoa is essential in cases of severe male infertility, especially those requiring surgical sperm retrieval. Although vitrifying individual spermatozoa on sperm vitrification devices (SpermVD®) provided optimal cell retrieval upon warming, motility rates tended to be lower than with bulk-freezing. Post-warming motility is directly affected by cryoprotectant exposure; however, optimal cryoprotectant equilibration time is unknown. We evaluated several timeframes exposing individual spermatozoa to cryoprotectant before freezing and different cryoprotectants. A total of 2,925 spermatozoa from 20 patients ranging from normozoospermic to moderate oligoteratoasthenozoospermic were vitrified in small groups, on 60 SpermVD®, in 1 µl droplets of 1:1 v/v cryoprotectant/washing medium mixture. Each group was vitrified after 2-60 minutes equilibration time. Motility of each group was evaluated after warming. Leftover pellets were frozen in cryotubes in a mixture of 1:1 v/v cryoprotectant/washing medium after 10 minutes equilibration at room temperature and 10 minutes on liquid nitrogen vapors. Post-thaw motility correlated negatively with cryoprotectant exposure time. The highest post-warming motility rate (32.1%) was observed with 8-minutes equilibration. After 10 minutes, motility rate of vitrified sperm was lower than that of bulk-freezing (31.7% vs. 37.0%, p < 0.0001). Different cryoprotectants did not affect the results. Therefore, for vitrifying small numbers of spermatozoa, we suggest maximum equilibration time of 8-minutes to achieve maximum motility after warming.

Heterogeneous expression of gamma-aminobutyric acid and gamma-aminobutyric acid-associated receptors and transporters in the rat suprachiasmatic nucleus.

The hypothalamic suprachiasmatic nucleus (SCN) is the primary mammalian circadian clock that regulates rhythmic physiology and behavior. The SCN is composed of a diverse set of neurons arranged in a tight intrinsic network. In the rat, vasoactive intestinal peptide (VIP)- and gastrin-releasing peptide (GRP)-containing neurons are the dominant cell phenotypes of the ventral SCN, and these cells receive photic information from the retina and the intergeniculate leaflet. Neurons expressing vasopressin (VP) are concentrated in the dorsal and medial aspects of the SCN. Although the VIP/GRP and VP cell groups are concentrated in different regions of the SCN, the separation of these cell groups is not absolute. The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is expressed in most SCN neurons irrespective of their location or peptidergic phenotype. In the present study, immunoperoxidase labeling, immunofluorescence confocal microscopy, and ultrastructural immunocytochemistry were used to examine the spatial distribution of several markers associated with SCN GABAergic neurons. Glutamate decarboxylase, a marker of GABA synthesis, and vesicular GABA transporter were more prominently observed in the ventral SCN. KCC2, a K(+)/Cl(-) cotransporter, was highly expressed in the ventral SCN in association with VIP- and GRP-producing neurons, whereas VP neurons in the dorsal SCN were devoid of KCC2. On the other hand, GABA(B) receptors were observed predominantly in VPergic neurons dorsally, whereas, in the ventral SCN, GABA(B) receptors were associated almost exclusively with retinal afferent fibers and terminals. The differential expression of GABAergic markers within the SCN suggests that GABA may play dissimilar roles in different SCN neuronal phenotypes.

Enhancement of glioma radiotherapy and chemotherapy response with targeted antibody therapy against death receptor 5.

PURPOSE: TRA-8 is an agonistic mouse monoclonal antibody that binds to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor 5, which induces apoptosis in cancer cells through a caspase-8-dependent mechanism. We investigated the ability of TRA-8 to augment the radiotherapy (RT) and chemotherapy response of human glioma cells in vitro and in vivo. METHODS AND MATERIALS: The in vitro cytotoxicity of TRA-8 and temozolomide (Tmz) or RT was examined using adenosine triphosphate-dependent viability and clonogenic survival assays with five glioma cell lines. Death receptor 5 expression was determined by flow cytometry. In vivo studies included subcutaneous and intracranial xenograft models testing various combination treatments, including RT, Tmz, and TRA-8. RESULTS: TRA-8, combined with Tmz or RT, produced enhanced cytotoxicity against five glioma cell lines compared with the use of the individual agents alone. Death receptor 5 upregulation occurred in response to RT. Complete tumor regression in the subcutaneous experiments was the most common in animals that received combination therapy with TRA-8/Tmz/RT. TRA-8 enhanced tumor growth delay in combination with RT or Tmz. TRA-8 alone had limited activity against intracranial tumors. In contrast, the median survival of mice treated with TRA-8/Tmz/RT was significantly greater than the control or TRA-8-alone-treated mice. The median survival of the mice treated with TRA-8/Tmz/RT or chemoradiotherapy only was significantly greater than the control or TRA-8-treated mice. A trend toward improved survival was observed between TRA-8/Tmz/RT-treated and Tmz/RT-treated mice. CONCLUSIONS: These preliminary findings support the hypothesis that TRA-8 will augment the RT and chemotherapy response in gliomas. A humanized version of TRA-8 is being evaluated in a Phase II clinical trial.

Fertility outcomes after extended searches for ejaculated spermatozoa in men with virtual azoospermia.

OBJECTIVE: To assess the fertility outcomes of extended searches for ejaculated spermatozoa in men with virtual azoospermia. DESIGN: A retrospective cohort of 242 couples whose male partner suffered from nonobstructive azoospermia and who were treated with the use of intracytoplasmic sperm injection (ICSI). SETTING: Not applicable. PATIENT(S): One hundred forty patients were referred to an extended search in the ejaculate and 102 patients were referred to microsurgical testicular sperm extraction (microTESE). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Rates of sperm retrieval, fertilization, and pregnancy, take-home baby rate, and missed abortion rate were analyzed and compared. RESULT(S): In the ejaculated spermatozoa group, motile spermatozoa were retrieved in 91 cases (65%) and on oocyte pick-up day in 71 cases (78%), compared with 70 cases (68%) in the microTESE group, with a similar incidence of sperm retrieval between groups. No significant difference was found between groups regarding mean number of embryo transfer and fertilization and pregnancy rates. There was no significant difference between groups regarding take-home baby rate. A significantly higher first-trimester missed abortion rate was found in the ejaculated sperm group (n = 14; 52%) compared with the microTESE group (n = 3; 8.6%). CONCLUSION(S): Conducting an extended spermatozoa search in the ejaculate of men with virtual azoospermia can provide pregnancy rates similar to those obtained with the use of microTESE, with a higher rate of spontaneous abortions in the ejaculate group.

Melanopsin retinal ganglion cells receive bipolar and amacrine cell synapses.

Melanopsin is a novel opsin synthesized in a small subset of retinal ganglion cells. Ganglion cells expressing melanopsin are capable of depolarizing in response to light in the absence of rod or cone input and are thus intrinsically light sensitive. Melanopsin ganglion cells convey information regarding general levels of environmental illumination to the suprachiasmatic nucleus, the intergeniculate leaflet, and the pretectum. Typically, retinal ganglion cells communicate information to central visual structures by receiving input from retinal photoreceptors via bipolar and amacrine cells. Because melanopsin ganglion cells do not require synaptic input to generate light-induced signals, these cells need not receive synapses from other neurons in the retina. In this study, we examined the ultrastructure of melanopsin ganglion cells in the mouse retina to determine the type (if any) of synaptic input these cells receive. Melanopsin immunoreaction product was associated primarily with the plasma membrane of (1) perikarya in the ganglion cell layer, (2) dendritic processes in the inner plexiform layer (IPL), and (3) axons in the optic fiber layer. Melanopsin-immunoreactive dendrites in the inner (ON) region of the IPL were postsynaptic to bipolar and amacrine terminals, whereas melanopsin dendrites stratifying in the outer (OFF) region of the IPL received only amacrine terminals. These observations suggested that rod and/or cone signals may be capable of modifying the intrinsic light response in melanopsin-expressing retinal ganglion cells.

Melanopsin mediates retrograde visual signaling in the retina.

The canonical flow of visual signals proceeds from outer to inner retina (photoreceptors → bipolar cells → ganglion cells). However, melanopsin-expressing ganglion cells are photosensitive and functional sustained light signaling to retinal dopaminergic interneurons persists in the absence of rods and cones. Here we show that the sustained-type light response of retinal dopamine neurons requires melanopsin and that the response is mediated by AMPA-type glutamate receptors, defining a retrograde retinal visual signaling pathway that fully reverses the usual flow of light signals in retinal circuits.

DING, a genistein target in human breast cancer: a protein without a gene.

Because most noncancer cells are tolerant to high micromolar concentrations of genistein (GEN), inhibitory or stimulatory effects of GEN have been claimed for a wide variety of biochemical targets that lead to a plethora of potential mechanisms. However, because GEN is present in tissues in the nanomol-per-liter range, most of these mechanisms are unlikely to be relevant in vivo. To better identify proteins that are targets of GEN, we used a GEN-agarose-affinity phase. Cytosols from human breast cancer MCF-7 cells were fractionated over a Sephadex diethylaminoethyl column, and nonabsorbed proteins in the flow-through were affinity absorbed onto a 2-carboxygenistein-agarose column. After proteins were washed with 100 mmol NaCl/L to remove weakly bound proteins, affinity elution was conducted with 1 mmol 2-carboxygenistein/L. Using this method, a p38 protein was recovered from MCF-7 cells. N-terminal chemical sequencing of the first 30 residues of the protein revealed a peptide sequence similar to those that have been discovered in human tissues (a T-cell attractant protein from synovial fluid from patients with osteoarthritis and an analogous human skin fibroblast protein using a hirudin-affinity column) as well as a cotonine-binding protein from rat brain and related proteins in plants. In each case, the corresponding gene has not been found. In conclusion, although much of the human genome has been sequenced, novel proteins that are not described by genome data remain to be found. The DING protein (N-terminal amino acid sequence Asp-Ile-Asn-Gly) that binds to genistein with high affinity is one of these. Its biological role, however, remains to be defined.