High-throughput screening method for discovering CatSper inhibitors using membrane depolarization caused by external calcium chelation and fluorescent cell barcoding

The exclusive expression of CatSper in sperm and its critical role in sperm function makes this channel an attractive target for contraception. The strategy of blocking CatSper as a male, non-hormonal contraceptive has not been fully explored due to the lack of robust screening methods to discover novel and specific inhibitors. The reason for this lack of appropriate methodology is the structural and functional complexity of this channel. We have developed a high-throughput method to screen drugs with the capacity to block CatSper in mammalian sperm. The assay is based on removing external free divalent cations by chelation, inducing CatSper to efficiently conduct monovalent cations. Since Na+ is highly concentrated in the extracellular milieu, a sudden influx depolarizes the cell. Using CatSper1 KO sperm we demonstrated that this depolarization depends on CatSper function. A membrane potential (Em) assay was combined with fluorescent cell barcoding (FCB), enabling higher throughput flow cytometry based on unique fluorescent signatures of different sperm samples. These differentially labeled samples incubated in distinct experimental conditions can be combined into one tube for simultaneous acquisition. In this way, acquisition times are highly reduced, which is essential to perform larger screening experiments for drug discovery using live cells. Altogether, a simple strategy for assessing CatSper was validated, and this assay was used to develop a high-throughput drug screening for new CatSper blockers.Fil: Luque, Guillermina Maria. 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: Schiavi Ehrenhaus, Liza Jamaica. 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: Jabloñski, Martina. 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: Balestrini, Paula Ania. 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: Novero, Analia Guadalupe. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Torres, Nicolás. 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: Osycka Salut, Claudia Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Darszon, Alberto. Universidad Autónoma del Estado de México; MéxicoFil: Krapf, Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Buffone, Mariano Gabriel. 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

Goodbye Hartmann trial: a prospective, international, multicenter, observational study on the current use of a surgical procedure developed a century ago

Background: Literature suggests colonic resection and primary anastomosis (RPA) instead of Hartmann's procedure (HP) for the treatment of left-sided colonic emergencies. We aim to evaluate the surgical options globally used to treat patients with acute left-sided colonic emergencies and the factors that leading to the choice of treatment, comparing HP and RPA. Methods: This is a prospective, international, multicenter, observational study registered on ClinicalTrials.gov. A total 1215 patients with left-sided colonic emergencies who required surgery were included from 204 centers during the period of March 1, 2020, to May 31, 2020. with a 1-year follow-up. Results: 564 patients (43.1%) were females. The mean age was 65.9 ± 15.6 years. HP was performed in 697 (57.3%) patients and RPA in 384 (31.6%) cases. Complicated acute diverticulitis was the most common cause of left-sided colonic emergencies (40.2%), followed by colorectal malignancy (36.6%). Severe complications (Clavien-Dindo ≥ 3b) were higher in the HP group (P < 0.001). 30-day mortality was higher in HP patients (13.7%), especially in case of bowel perforation and diffused peritonitis. 1-year follow-up showed no differences on ostomy reversal rate between HP and RPA. (P = 0.127). A backward likelihood logistic regression model showed that RPA was preferred in younger patients, having low ASA score (≤ 3), in case of large bowel obstruction, absence of colonic ischemia, longer time from admission to surgery, operating early at the day working hours, by a surgeon who performed more than 50 colorectal resections. Conclusions: After 100 years since the first Hartmann's procedure, HP remains the most common treatment for left-sided colorectal emergencies. Treatment's choice depends on patient characteristics, the time of surgery and the experience of the surgeon. RPA should be considered as the gold standard for surgery, with HP being an exception

Vestibular mapping in patients with unilateral peripheral-vestibular deficits.

OBJECTIVE To test the hypothesis that patterns of semicircular canal (SCC) and otolith impairment in unilateral vestibular-loss depend on the underlying disorders, we analyzed peripheral-vestibular function of all five vestibular sensors. METHODS Retrospective case-series. Screening of the hospital video-head-impulse test database (n=4983) for patients with unilaterally impaired SCC-function who also received ocular vestibular-evoked myogenic-potentials and cervical vestibular-evoked myogenic-potentials (n=302). Frequency of impairment of vestibular end-organs (horizontal/anterior/posterior SCC, utriculus/sacculus) was analyzed with hierarchical cluster-analysis and correlated with the underlying etiology. RESULTS Acute vestibular-neuropathy (AVN) (37.4%, 113/302), vestibular schwannoma (18.2%, 55/302) and acute cochleo-vestibular neuropathy (6.6%, 20/302) were most frequent. Horizontal SCC-impairment (87.4%, 264/302) was more frequent (p<0.001) than posterior (47.4%, 143/302) and anterior (37.8 %, 114/302) SCC-impairment. Utricular damage (58%, 175/302) was noted more often (p=0.003) than saccular impairment (32%, 98/302). On average 2.6 (95%-CI=2.48-2.78) vestibular sensors were deficient, with higher numbers (p≤0.017) for acute cochleo-vestibular neuropathy and vestibular schwannoma than for AVN, Menière's disease and episodic-vestibular-syndrome. In hierarchical cluster-analysis, early mergers (posterior SCC/sacculus; anterior SCC/utriculus) pointed to closer pathophysiological association of these sensors, whereas the late merger of the horizontal canal indicated a more distinct state. CONCLUSIONS While the extent and pattern of vestibular impairment critically depended on the underlying disorder, more limited damage in AVN and Menière's disease was noted, emphasizing the individual range of loss-of-function and the value of vestibular-mapping. Likely, both the anatomical properties of the different vestibular end-organs and their vulnerability to external factors contribute to the relative sparing of the vertical canals and the sacculus

Pre-habilitation Before Vestibular Schwannoma Surgery—Impact of Intratympanal Gentamicin Application on the Vestibulo-Ocular Reflex

Background: Patients with vestibular schwannoma that show residual peripheral-vestibular function before surgery may experience sudden and substantial vestibular loss of function after surgical resection. To alleviate the sudden loss of peripheral-vestibular function after vestibular-schwannoma (VS) resection, pre-surgical intratympanic gentamicin application was proposed.Objective: We hypothesized that this approach allows for a controlled reduction of peripheral-vestibular function before surgery but that resulting peripheral-vestibular deficits may be canal-specific with anterior-canal sparing as observed previously in systemic gentamicin application.Methods: Thirty-four patients (age-range = 27–70 y) with unilateral VS (size = 2–50 mm) were included in this retrospective single-center trial. The angular vestibulo-ocular reflex (aVOR) was quantified before and after (29.7 ± 18.7 d, mean ± 1SD) a single or two sequential intratympanic gentamicin applications by use of video-head-impulse testing. Both aVOR gains, cumulative saccadic amplitudes, and overall aVOR function were retrieved. Statistical analysis was done using a generalized linear model.Results: At baseline, loss of function of the horizontal (20/34) and posterior (21/34) canal was significantly (p &lt; 0.001) more frequent than that of the anterior canal (5/34). After gentamicin application, loss of function of the horizontal (32/34) or posterior (31/34) canal remained significantly (p ≤ 0.003) more frequent than that of the anterior canal (18/34). For all ipsilesional canals, significant aVOR-gain reductions and cumulative-saccadic-amplitude increases were noted after gentamicin. For the horizontal canal, loss of function was significantly larger (increase in cumulative-saccadic-amplitude: 1.6 ± 2.0 vs. 0.8 ± 1.2, p = 0.007) or showed a trend to larger changes (decrease in aVOR-gain: 0.24 ± 0.22 vs. 0.13 ± 0.29, p = 0.069) than for the anterior canal.Conclusions: Intratympanic gentamicin application resulted in a substantial reduction in peripheral-vestibular function in all three ipsilesional canals. Relative sparing of anterior-canal function noted at baseline was preserved after gentamicin treatment. Thus, pre-surgical intratympanic gentamicin is a suitable preparatory procedure for reducing the drop in peripheral-vestibular function after VS-resection. The reasons for relative sparing of the anterior canal remain unclear

Hierarchical Cluster Analysis of Semicircular Canal and Otolith Deficits in Bilateral Vestibulopathy

Background Gait imbalance and oscillopsia are frequent complaints of bilateral vestibular loss (BLV). Video-head-impulse testing (vHIT) of all six semicircular canals (SCCs) has demonstrated varying involvement of the different canals. Sparing of anterior-canal function has been linked to aminoglycoside-related vestibulopathy and Menière's disease. We hypothesized that utricular and saccular impairment [assessed by vestibular-evoked myogenic potentials (VEMPs)] may be disease-specific also, possibly facilitating the differential diagnosis. Methods We searched our vHIT database ( = 3,271) for patients with bilaterally impaired SCC function who also received ocular VEMPs (oVEMPs) and cervical VEMPs (cVEMPs) and identified 101 patients. oVEMP/cVEMP latencies above the 95th percentile and peak-to-peak amplitudes below the 5th percentile of normal were considered abnormal. Frequency of impairment of vestibular end organs (horizontal/anterior/posterior SCC, utriculus/sacculus) was analyzed with hierarchical cluster analysis and correlated with the underlying etiology. Results Rates of utricular and saccular loss of function were similar (87.1 vs. 78.2%,  = 0.136, Fisher's exact test). oVEMP abnormalities were found more frequent in aminoglycoside-related bilateral vestibular loss (BVL) compared with Menière's disease (91.7 vs. 54.6%,  = 0.039). Hierarchical cluster analysis indicated distinct patterns of vestibular end-organ impairment, showing that the results for the same end-organs on both sides are more similar than to other end-organs. Relative sparing of anterior-canal function was reflected in late merging with the other end-organs, emphasizing their distinct state. An anatomically corresponding pattern of SCC/otolith hypofunction was present in 60.4% (oVEMPs vs. horizontal SCCs), 34.7% (oVEMPs vs. anterior SCCs), and 48.5% (cVEMPs vs. posterior SCCs) of cases. Average (±1 SD) number of damaged sensors was 6.8 ± 2.2 out of 10. Significantly ( < 0.001) more sensors were impaired in patients with aminoglycoside-related BVL (8.1 ± 1.2) or inner-ear infections (8.7 ± 1.8) compared with Menière-related BVL (5.5 ± 1.5). Discussion Hierarchical cluster analysis may help differentiate characteristic patterns of BVL. With a prevalence of ≈80%, utricular and/or saccular impairment is frequent in BVL. The extent of SCC and otolith impairment was disease-dependent, showing most extensive damage in BVL related to inner-ear infection and aminoglycoside-exposure and more selective impairment in Menière's disease. Specifically, assessing utricular function may help in the distinction between aminoglycoside-related BVL and bilateral Menière's disease

Distinct Vestibular Evoked Myogenic Potentials in Patients With Parkinson Disease and Progressive Supranuclear Palsy

Background: Early brainstem neurodegeneration is common in Parkinson's disease (PD) and progressive supranuclear palsy (PSP). While previous work showed abnormalities in vestibular evoked myogenic potentials (VEMPs) in patients with either disorder as compared to healthy humans, it remains unclear whether ocular and cervical VEMPs differ between PD and PSP patients.Methods: We prospectively included 12 PD and 11 PSP patients, performed ocular and cervical VEMPs, and calculated specific VEMP scores (0 = normal, 12 = most pathological) based on latencies, amplitude, and absent responses. In addition, we assessed disease duration, presence of imbalance, motor asymmetry, and motor disability using the Movement Disorder Society Unified Parkinson's Disease Rating Scale, part III (MDS-UPDRS III). Moreover, we ascertained various sleep parameters by video-polysomnography.Results: PSP and PD patients had similar oVEMP scores (6 [3–6] vs. 3 [1.3–6], p = 0.06), but PSP patients had higher cVEMP scores (3 [0–6] vs. 0 [0–2.8], p = 0.03) and total VEMP scores (9 [5–12] vs. 4 [2–7.5], p = 0.01). Moreover, total VEMP scores &gt;10 were only observed in PSP patients (45%, p = 0.01). MDS-UPDRS III correlated with cVEMP scores (rho = 0.77, p = 0.01) in PSP, but not in PD. In PD, but not in PSP, polysomnographic markers of disturbed sleep, including decreased rapid eye movement sleep, showed significant correlations with VEMP scores.Conclusions: Our findings suggest that central vestibular pathways are more severely damaged in PSP than in PD, as indicated by higher cervical and total VEMP scores in PSP than PD in a between-groups analysis. Meaningful correlations between VEMPs and motor and non-motor symptoms further encourage its use in neurodegenerative Parkinsonian syndromes

Hierarchical Cluster Analysis of Semicircular Canal and Otolith Deficits in Bilateral Vestibulopathy

BackgroundGait imbalance and oscillopsia are frequent complaints of bilateral vestibular loss (BLV). Video-head-impulse testing (vHIT) of all six semicircular canals (SCCs) has demonstrated varying involvement of the different canals. Sparing of anterior-canal function has been linked to aminoglycoside-related vestibulopathy and Menière’s disease. We hypothesized that utricular and saccular impairment [assessed by vestibular-evoked myogenic potentials (VEMPs)] may be disease-specific also, possibly facilitating the differential diagnosis.MethodsWe searched our vHIT database (n = 3,271) for patients with bilaterally impaired SCC function who also received ocular VEMPs (oVEMPs) and cervical VEMPs (cVEMPs) and identified 101 patients. oVEMP/cVEMP latencies above the 95th percentile and peak-to-peak amplitudes below the 5th percentile of normal were considered abnormal. Frequency of impairment of vestibular end organs (horizontal/anterior/posterior SCC, utriculus/sacculus) was analyzed with hierarchical cluster analysis and correlated with the underlying etiology.ResultsRates of utricular and saccular loss of function were similar (87.1 vs. 78.2%, p = 0.136, Fisher’s exact test). oVEMP abnormalities were found more frequent in aminoglycoside-related bilateral vestibular loss (BVL) compared with Menière’s disease (91.7 vs. 54.6%, p = 0.039). Hierarchical cluster analysis indicated distinct patterns of vestibular end-organ impairment, showing that the results for the same end-organs on both sides are more similar than to other end-organs. Relative sparing of anterior-canal function was reflected in late merging with the other end-organs, emphasizing their distinct state. An anatomically corresponding pattern of SCC/otolith hypofunction was present in 60.4% (oVEMPs vs. horizontal SCCs), 34.7% (oVEMPs vs. anterior SCCs), and 48.5% (cVEMPs vs. posterior SCCs) of cases. Average (±1 SD) number of damaged sensors was 6.8 ± 2.2 out of 10. Significantly (p &lt; 0.001) more sensors were impaired in patients with aminoglycoside-related BVL (8.1 ± 1.2) or inner-ear infections (8.7 ± 1.8) compared with Menière-related BVL (5.5 ± 1.5).DiscussionHierarchical cluster analysis may help differentiate characteristic patterns of BVL. With a prevalence of ≈80%, utricular and/or saccular impairment is frequent in BVL. The extent of SCC and otolith impairment was disease-dependent, showing most extensive damage in BVL related to inner-ear infection and aminoglycoside-exposure and more selective impairment in Menière’s disease. Specifically, assessing utricular function may help in the distinction between aminoglycoside-related BVL and bilateral Menière’s disease