Predicting the functional states of human iPSC-derived neurons with single-cell RNA-seq and electrophysiology.

Human neural progenitors derived from pluripotent stem cells develop into electrophysiologically active neurons at heterogeneous rates, which can confound disease-relevant discoveries in neurology and psychiatry. By combining patch clamping, morphological and transcriptome analysis on single-human neurons in vitro, we defined a continuum of poor to highly functional electrophysiological states of differentiated neurons. The strong correlations between action potentials, synaptic activity, dendritic complexity and gene expression highlight the importance of methods for isolating functionally comparable neurons for in vitro investigations of brain disorders. Although whole-cell electrophysiology is the gold standard for functional evaluation, it often lacks the scalability required for disease modeling studies. Here, we demonstrate a multimodal machine-learning strategy to identify new molecular features that predict the physiological states of single neurons, independently of the time spent in vitro. As further proof of concept, we selected one of the potential neurophysiological biomarkers identified in this study-GDAP1L1-to isolate highly functional live human neurons in vitro.

Dual effects of α2 -adrenoceptors in modulating myogenic tone in sheep isolated internal anal sphincter.

BACKGROUND: The role of α-adrenoceptors in promoting continence through modulation of sphincter tone has focused primarily on the effects of α1 -adrenoceptors. We have used three clinically available agents, which are selective for α2 -adrenoceptors, to investigate their role in contractile and neurogenic responses on the internal anal sphincter (IAS). METHODS: IAS strips, which had spontaneously generated tone, were used to investigate the contractile effect of lofexidine, brimonidine, and dexmedetomidine on muscle tone in the presence or absence of subtype selective antagonists. The effect of brimonidine on the magnitude and time course of neurogenic responses generated by electrical field stimulation (EFS) was also examined. The affinity of test compounds at α1 - and α2 -adrenoceptors was established by competition binding with [3H]-prazosin and [3H]-RX821002. KEY RESULTS: All agonists caused concentration-dependent contraction of the IAS and lofexidine demonstrated an enantiomeric difference in potency with a 10-fold difference between the (-) and (+) isomers. Responses to lofexidine and dexmedetomidine were inhibited in the presence of the α1 -adrenoceptor selective antagonist prazosin, but not in the presence of RX811059 (α2 -adrenoceptor selective antagonist); brimonidine responses were inhibited by RX811059 and, to a lesser extent, by prazosin. Brimonidine affected both magnitude and duration of neurogenic responses, which was reversed in the presence of RX811059. CONCLUSIONS & INFERENCES: We conclude that α2 -adrenoceptors can mediate contraction of IAS, although this effect is most evident with efficacious imidazoline agonists rather than the most selective ligand. In addition, this receptor subtype can directly inhibit noradrenergic contractile responses to EFS and, indirectly, enhance nitrergic relaxatory responses.

[Cutaneous side effects of the multikinase inhibitors sorafenib and sunitinib]. / Kutane Nebenwirkungen der Multikinaseinhibitoren Sorafenib und Sunitinib.

Molecular targeted therapy with monoclonal antibodies and low molecular weight inhibitors is gaining increasing importance particularly in the treatment of malignant tumors. These drugs are specific and highly selective agents that intervene in the dysfunctional regulatory processes of malignant cells in order to influence cell proliferation, cell differentiation, or angiogenesis. The multikinase inhibitors sorafenib and sunitinib exhibit a favorable tolerability profile and spectrum of side effects, especially in comparison to conventional chemotherapeutic agents. However, they induce specific cutaneous side effects that can in turn be indicators of antitumor activity. This review article compares the cutaneous side effects of the two multikinase inhibitors sorafenib and sunitinib and discusses the therapeutic options for the individual cutaneous reaction patterns.

Investigation of the distribution and function of alpha-adrenoceptors in the sheep isolated internal anal sphincter.

BACKGROUND AND PURPOSE: We have investigated the distribution of alpha-adrenoceptors in sheep internal anal sphincter (IAS), as a model for the human tissue, and evaluated various imidazoline derivatives for potential treatment of faecal incontinence. EXPERIMENTAL APPROACH: Saturation and competition binding with (3)H-prazosin and (3)H-RX821002 were used to confirm the presence and density of alpha-adrenoceptors in sheep IAS, and the affinity of imidazoline compounds at these receptors. A combination of in vitro receptor autoradiography and immunohistochemistry was used to investigate the regional distribution of binding sites. Contractile activity of imidazoline-based compounds on sheep IAS was assessed by isometric tension recording. KEY RESULTS: Saturation binding confirmed the presence of both alpha(1)- and alpha(2)-adrenoceptors, and subsequent characterization with sub-type-selective agents, identified them as alpha(1A)- and alpha(2D)-adrenoceptor sub-types. Autoradiographic studies with (3)H-prazosin showed a positive association of alpha(1)-adrenoceptors with immunohistochemically identified smooth muscle fibres. Anti-alpha(1)-adrenoceptor immunohistochemistry revealed similar distributions of the receptor in sheep and human IAS. The imidazoline compounds caused concentration-dependent contractions of the anal sphincter, but the maximum responses were less than those elicited by l-erythro-methoxamine, a standard non-imidazoline alpha(1)-adrenoceptor agonist. Prazosin (selective alpha(1)-adrenoceptor antagonist) significantly reduced the magnitude of contraction to l-erythro-methoxamine at the highest concentration used. Both prazosin and RX811059 (a selective alpha(2)-adrenoceptor antagonist) reduced the potency (pEC(50)) of clonidine. CONCLUSIONS AND IMPLICATIONS: This study shows that both alpha(1)- and alpha(2)-adrenoceptors are expressed in the sheep IAS, and contribute (perhaps synergistically) to contractions elicited by various imidazoline derivatives. These agents may prove useful in the treatment of faecal incontinence.

Microbiological analysis of epidermal growth factor receptor inhibitor therapy-associated paronychia.

BACKGROUND: Paronychia is a well-known, but difficult to treat cutaneous toxicity associated with epidermal growth factor receptor (EGFR) inhibitor therapy. Although bacterial and fungal infections as well as mechanical trauma may play a role as co-pathogens, there is no good basis for an empirical antimicrobial chemotherapy in these patients. MATERIALS AND METHODS: We retrospectively analysed the microbiological results and resistance analysis of 42 cases of EGFR inhibitor-associated paronychia induced by cetuximab. RESULTS: We identified 20 different species, among these 72% Gram-positive bacteria, 23% Gram-negative bacteria and 5%Candida species. About half of the microbes identified may be considered as residential bacterial flora of the skin, but isolation of microbes from paronychia may indicate a pathogenic relevance for this type of reaction. Eight of our patients were treated with oral antibiotics, whereas two patients received oral antimycotic therapy. All other cases of paronychia were controlled using topical antiseptic, antibiotic and antimycotic agents. CONCLUSION: Empirical oral antibiotic treatment may be performed with oral cephalosporines, ciprofloxacin, levofloxacin or moxifloxacin, as these antimicrobials have high in vitro activity against the majority of the isolated microorganisms and reach high concentrations in the relevant tissue.

Investigation of the role of adrenergic and non-nitrergic, non-adrenergic neurotransmission in the sheep isolated internal anal sphincter.

Nitric oxide is widely established as an important neurotransmitter in the control of anal sphincter tone; although, a number of other transmitters have also been tentatively implicated. Whilst alpha-adrenoceptor antagonists reduce anal sphincter pressure in man, the role of noradrenaline as a possible transmitter is poorly characterised. We have investigated the contribution of these transmitters to neurogenic relaxations, and evaluated the possible role of a non-nitrergic, non-adrenergic transmitter. The magnitude and duration of neurogenic responses were examined by measuring responses to electrical field stimulation (EFS) in segments of sheep internal anal sphincter following the development of spontaneous myogenic tone. Neurogenic relaxations induced by EFS were significantly reduced in the presence of N(G)-nitro-L-arginine methyl ester (L-NAME) suggesting major involvement of nitric oxide as a neurotransmitter. The duration of neurogenic relaxations was inversely related to the frequency of EFS, with contractile responses often manifest at higher frequencies. The duration of relaxations at high frequencies of EFS was increased by bretylium (adrenergic neurone blocker) and prazosin (alpha(1)-adrenoceptor antagonist). At higher frequencies of EFS, 60% of preparations also produced a residual non-nitrergic, non-adrenergic, apamin-sensitive relaxation which was unaffected by vasoactive intestinal polypeptide (VIP) and inhibitors of purinergic responses [suramin, pyridoxal-phosphate-6-azophenyl 2',4' disulfonic acid (PPADS) and alpha,beta-methylene adenosine triphosphate (ATP)]. However, MRS2179 (P2Y(1) receptor antagonist) showed a modest inhibitory effect. We conclude that endogenous noradrenaline acts via postjunctional alpha(1)-adrenoceptors to antagonize neurogenic relaxations that are largely mediated by nitric oxide. Our results indicate the involvement of a non-nitrergic, non-adrenergic, apamin-sensitive transmitter which is inhibited by MRS2179, suggesting a possible role for purines.

Interpreting legal mandates. Assistance dogs in medical facilities.

Many disabled persons who visit or work in medical facilities have been denied the right to be accompanied by their guide, hearing and service dogs. Guidelines that meet legal mandates must be based on realistic assessments and a correct interpretation of the law.

Energetics of 3-oxo-delta 5-steroid isomerase: source of the catalytic power of the enzyme.

Knowledge of the partitioning of the putative dienol intermediate (2) by steroid isomerase (KSI) (Hawkinson et al. 1991), in conjunction with various steady-state kinetic parameters, allows elucidation of the detailed free energy profile for the KSI-catalyzed conversion of 5-androstene-3,17-dione (1) to 4-androstene-3,17-dione (3). This free energy profile shows four kinetically significant energy barriers (substrate binding, the two chemical steps, and dissociation of product) that must be traversed upon conversion of 1 to 3. Thus, no single step of the catalytic cycle is cleanly rate-limiting. The source of the catalytic power of KSI is discussed via comparison of the free energy profile for the KSI-catalyzed isomerization with those for the acetate-catalyzed isomerization and the aqueous reaction at pH 7. Similarities between the energetics of the KSI-catalyzed and triosephosphate isomerase catalyzed reactions are also noted.

Kinetic competence of an externally generated dienol intermediate with steroid isomerase.

The putative intermediate dienol (2) in the steroid isomerase (KSI) catalyzed conversion of 5-androstene-3,17-dione (1) to 4-androstene-3,17-dione (3) has been independently generated and tested as a substrate for KSI. At pH 7, dienol 2 is converted by KSI to a mixture of 1 (46%) and 3 (54%). The apparent second-order rate constant for reaction of 2 with KSI to produce 3 (kappa cat/Km = 2.3 x 10(8) M-1 s-1) is similar to that for reaction of 1 with KSI (kappa cat/Km = 2.1 x 10(8) M-1 s-1), demonstrating that 2 is kinetically competent. Isomerization of 1 by KSI in D2O gives only 5% of solvent deuterium incorporated into the product 3. When 2 reacts with KSI in D2O, and the product 3 is isolated (from direct reaction of 2 and from subsequent conversion of the 1 initially formed), ca. 80 atom % deuterium is located at C-6 beta, confirming that protonation of the dienol by KSI occurs at the same face as the proton transfer in the KSI catalyzed reaction of 1 to 3.

Orientation, accessibility, and mobility of equilenin bound to the active site of steroid isomerase.

The fluorescent aromatic steroid equilenin, which contains a beta-naphthol moiety, is bound by 3-oxo-delta 5-steroid isomerase. The excitation and emission fluorescence spectra of equilenin when bound to the enzyme, as well as the fluorescence decay time, are indicative of ground-state ionization. In view of the high efficiency of tyrosine quenching, which approaches 100%, the beta-naphthol moiety of equilenin must be in proximity to all three tyrosines of steroid isomerase to account for the observed efficiency of radiationless energy transfer. From the observed response to an external quencher, it appears that enzyme-bound equilenin is largely shielded from solvent. Fluorescence anisotropy measurements indicate a high degree of immobilization of the bound ligand. These models are consistent with proposed models of the enzyme-substrate complex.