Early-onset encephalomyopathy associated with tissue-specific mitochondrial DNA depletion: A morphological,biochemical and molecular-genetic study

A male infant, born from consanguineous parents, suffered from birth with a progressive neuromuscular disorder characterized by psychomotor delay, hypotonia, muscle weakness and wasting, deep-tendon areflexia and spastic posture. High levels of lactic acid in blood and cerebrospinal fluid suggested a mitochondrial respiratory chain defect. Muscle biopsy revealed raggedred and cytochromec oxidase-negative fibres, lipid accumulation and dystrophic changes. Multiple defects of respiratory complexes were detected in muscle homogenate, but cultured fibroblasts, myoblasts and myotubes were normal. Southern blot analysis showed markedly reduced levels of mitochondrial DNA (mtDNA) in muscle, while lymphocytes, fibroblasts and muscle precursor cells were normal. Neither depletion of mtDNA nor abnormalities of the respiratory complexes were observed in innervated muscle fibres cultured for as long as 4 months. No mutations were observed in two candidate nuclear genes,mtTFA andmtSSB, retro-transcribed, amplified and sequenced from the proband's mRNA. Sequence analysis of the mtDNA D-loop and of the origin of replication of the mtDNA light strand failed to identify potentially pathogenic mutations of these replicative elements in the proband's muscle mtDNA. Our findings indicate that mtDNA depletion is due to a nuclear encoded gene and suggest that the abnormality underlying defective mtDNA propagation must occur after muscle differentiation in vivo.     

Expression of Epstein-Barr virus latent membrane protein-1 in Hodgkin and Reed-Sternberg cells of classical Hodgkin's lymphoma: associations with presenting features, serum interleukin 10 levels, and clinical outcome.

PURPOSE: EBV-latent membrane protein-1 (LMP-1) is often expressed in Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin's lymphoma (cHL), but its clinical significance is controversial. We correlated LMP-1 with presenting features, including serum interleukin 10 levels and clinical outcome. EXPERIMENTAL DESIGN: Patients were eligible if they had biopsy-proven cHL, were untreated, HIV-1 negative, and had available archival tissue. LMP-1 expression was determined by immunohistochemistry. RESULTS: We identified 577 patients with cHL with a median age of 30 years, 55% of whom were male. LMP-1 was expressed in HRS cells of 124 patients (21%) and was detected in 78 of 461 (17%) patients with nodular sclerosis compared with 44 of 112 (39%) with mixed cellularity (P < 0.001 by Fisher's exact test). Patients with tumors with LMP-1-positive HRS cells had higher serum interleukin 10 levels (P = 0.009 by Mann-Whitney test). For the 303 patients treated with doxorubicin, bleomycin, vinblastine, and dacarbazine or equivalent regimens, the 5-year failure-free survival (FFS) for those with LMP-1-positive tumors was 74% compared with 81% for those with LMP-1-negative tumors (P = 0.23, by log-rank test). Overall survival (OS) at 5 years for patients with LMP-1-positive tumors was 90 versus 91% for patients with LMP-1-negative tumors (P = 0.8 by log-rank test). Expression of LMP-1 was not associated with different FFS and OS in patients treated with other regimens or with radiotherapy alone. CONCLUSIONS: LMP-1 was expressed by HRS cells in 21% of cHL and correlated with mixed cellularity type and higher serum interleukin 10 levels. The presence of LMP-1 was not associated with FFS or OS in uniformly treated patients.     

Glycolipids from sponges. Part 17. Clathrosides and isoclathrosides, unique glycolipids from the Caribbean sponge Agelas clathrodes

Two families of unique glycolipids, clathrosides A-C (2a-4a) and isoclathrosides A-C (5a-7a) were isolated from the Caribbean sponge Agelas clathrodes. Clathrosides and isoclathrosides are glycosides of a very-long-chain alcohol derived from fatty acids, a new class of glycolipids that appears to be characteristic of marine sponges. The six compounds differ in configuration and in the branching of alkyl chains. Stereostructures of the clathrosides were determined by NMR and CD spectroscopy, mass spectrometry, and chemical degradation. Location of the methyl branch on the proper alkyl chain required an exceptional 1-D TOCSY experiment, in which coherence was transferred through as many as 13 vicinal couplings.     

Assessment of Angiogenesis and Cell Survivability of an Inkjet Bioprinted Biological Implant in an Animal Model

The rapidly growing field of tissue engineering hopes to soon address the shortage of transplantable tissues, allowing for precise control and fabrication that could be made for each specific patient. The protocols currently in place to print large-scale tissues have yet to address the main challenge of nutritional deficiencies in the central areas of the engineered tissue, causing necrosis deep within and rendering it ineffective. Bioprinted microvasculature has been proposed to encourage angiogenesis and facilitate the mobility of oxygen and nutrients throughout the engineered tissue. An implant made via an inkjet printing process containing human microvascular endothelial cells was placed in both B17-SCID and NSG-SGM3 animal models to determine the rate of angiogenesis and degree of cell survival. The implantable tissues were made using a combination of alginate and gelatin type B; all implants were printed via previously published procedures using a modified HP inkjet printer. Histopathological results show a dramatic increase in the average microvasculature formation for mice that received the printed constructs within the implant area when compared to the manual and control implants, indicating inkjet bioprinting technology can be effectively used for vascularization of engineered tissues.     

Semen processing by density gradient centrifugation does not improve sperm apoptotic deoxyribonucleic acid fragmentation rates

We wished to verify whether semen processing by discontinuous double-layered density gradient centrifugation could improve sperm apoptotic DNA fragmentation rates using a commercially available deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay in 35 consecutive men presenting for assisted reproductive treatments. Although sperm motility did improve as expected, no effects were observed in sperm apoptotic DNA fragmentation rates, and this should be considered in the routine assisted reproduction setting.     

The risk of shoulder dystocia related permanent fetal injury in relation to birth weight

OBJECTIVE: To examine birth weight related risks of fetal injury in connection with shoulder dystocia. STUDY DESIGN: The investigation was based on a retrospective analysis of 316 fetal neurological injuries associated with deliveries complicated by arrest of the shoulders that occurred across the United States. RESULTS: The study revealed that the distribution of birthweights for the high risk shoulder dystocia population differs from the standard birthweight distribution. The relative difference per birthweight interval is used to adjust an assumed 1:1000 baseline risk of injury due to shoulder dystocia following vaginal deliveries. These adjusted risks show a need to consider new thresholds for elective cesarean delivery. CONCLUSIONS: Current North American and British guidelines, that set 5000 g as minimum estimated fetal weight limit for elective cesarean section in non-diabetic and 4500 g for diabetic gravidas, may expose some macrosomic fetuses to a high risk of permanent neurological damage. The authors present the opinion that the mother, having been informed of the risks of vaginal versus abdominal delivery, should be allowed to play an active role in the critical management decisions.     

Accidental ingestion of sodium molybdate at the workplace followed by short-term biomonitoring

Introduction:Most of the molybdenum (Mo) is used in metallurgical applications, the tetrathiomolybdate form is an experimental chelating agent for Wilson’s disease. Human data of acute Mo exposure are lacking and, no report of no-observed-adverse-effect level (NOAEL) has been described until now.Case study:We report a case of acute occupational exposure to molybdenum, with the related plasma and urine molybdenum concentrations, caused by an accidental ingestion of a sip of an anti-corrosion liquid for metal containing sodium molybdate. Our purpose was to evaluate potential systemic toxicity of molybdenum and to evaluate the dose-response/dose-effect relationship. We estimated the amount of ingested molybdenum to make a mg/kg relationship and performed repeated urine and plasma molybdenum determinations. The patient was hospitalized for three days to monitor possible development of acute symptoms/biochemical alterations.Discussion:We estimated the amount of the sip around 50 ml, with an estimation of a total of 5 gr of sodium molybdate that, for the patient bodyweight of 80 kg, would mean 62,5 mg/kg of ingested Mo. Blood and urine samples collected 2 hours after ingestion showed 50 mcg/L (reference range: 0.43 – 1.8 mcg/L) and 630 mcg/L (refence range: up to 116 mcg/L) of Mo respectively, confirming acute exposure. The patients remained asymptomatic confirming that an estimated oral dose of Mo of 62.5 mg/kg was not associated with adverse effects.Conclusions:Our value, being extrapolated by a single case, will require further confirmations from other studies to allow a full evaluation of a NOAEL. Nevertheless, it does not preclude its use in evaluating the probable absence of adverse effect in the context of acute Mo exposure.     

Mechanism of voltage sensing in Ca2+- and voltage-activated K+ (BK) channels

In neurosecretion, allosteric communication between voltage sensors and Ca²⁺ binding in BK channels is crucially involved in damping excitatory stimuli. Nevertheless, the voltage-sensing mechanism of BK channels is still under debate. Here, based on gating current measurements, we demonstrate that two arginines in the transmembrane segment S4 (R210 and R213) function as the BK gating charges. Significantly, the energy landscape of the gating particles is electrostatically tuned by a network of salt bridges contained in the voltage sensor domain (VSD). Molecular dynamics simulations and proton transport experiments in the hyperpolarization-activated R210H mutant suggest that the electric field drops off within a narrow septum whose boundaries are defined by the gating charges. Unlike Kv channels, the charge movement in BK appears to be limited to a small displacement of the guanidinium moieties of R210 and R213, without significant movement of the S4.

Excitable tissues accomplish their signaling functions thanks in part to the interplay of several voltage-sensitive ion channels (1–6). Hence, to understand these processes, it is crucial to establish how voltage-sensitive ion channels sense changes in the electric field across the membrane, an issue that has been a matter of extensive study and intense debate for decades. The most widely accepted mechanism proposes the existence of voltage-sensor domains (VSDs), modules that undergo two or more discrete conformational states in response to changes in the membrane voltage. The simplest model considers two states: active (A), which promotes pore opening, and resting (R), which promotes channel closing. To accomplish its function, VSDs contain voltage-sensitive particles, which move in response to changes in the electric field. This movement triggers the interconversion between the two discrete conformational states. These voltage-sensing particles are typically the guanidine groups of arginine residues within the S4 transmembrane segment, which undergo a combination of rotational, translational, and tilting movement in response to changes in membrane voltage (7–14).The large-conductance Ca²⁺- and voltage-activated K⁺ (BK) channels have a wide distribution in mammalian tissues (15–18), where they participate in a diversity of physiological processes. Their malfunction is often related to diverse pathological conditions (19, 20). BK channel open probability is independently regulated by membrane depolarization and intracellular Ca²⁺ concentration (21, 22), each stimulus being detected by specialized modules. Like other voltage-sensitive K⁺ (Kv) channels, BK is an homotetramer in which each of its α subunits consists of a pore domain (PD; S5-S6 transmembrane segments), a voltage-sensing domain (VSD; S1–S4 transmembrane segments) containing a positively charged S4, and a cytosolic C-terminal regulatory domain, which contains the Ca²⁺-binding sites (23, 24). Also, like some members of other K⁺ channel families (25, 26), the VSD and PD of BK are non–domain swapped (23, 24). BK channels display some distinctive structural and functional features: Despite sharing the selectivity filter sequence with Kv channels, BK unitary conductance and selectivity are exquisitely high (27–30). The BK α subunit has an additional transmembrane segment S0 [therefore, its N terminus faces the extracellular medium (31)], and the voltage sensitivity in BK channels is significantly lower than that of Kv channels, presumably because of their lower number of gating charges (32).Although thoroughly studied, research into BK VSD and its voltage dependence has faced several technical obstacles. The relatively small gating charge per channel (32) and the large conductance of the BK pore makes isolating of the gating currents from the ionic currents a tough experimental challenge. In addition, because mutations of VSD residues can produce very large shifts in both the gating charge-voltage (Q(V)) and the conductance-voltage G(V)) relationships (33), it is necessary to use extreme voltages to accurately measure the voltage dependence of some mutants. Consequently, the identification of BK gating charges has been addressed by using indirect approaches (33, 34). The combination of electrophysiology measurements and kinetic modeling suggests a decentralized VSD in the BK channel, where four charged residues (D153 and R167 in S2, D186 in S3, and R213 in S4) act as voltage sensor particles (33). A recent report of the atomistic cryo-electron microscopy (cryo-EM) structures of the human BK channel and its homolog in Aplysia californica (AcSlo) revealed minor structural differences between the VSD in both the Ca²⁺-bound (open pore) and the Ca²⁺-unbound (closed pore) conformations (23, 24, 35). This result can be explained if the conformational changes of the BK VSD upon activation are small compared to those that occur during the activation of other channels, such as HCN channels (12–14).In this study, we identified voltage-sensing particles in the BK channel by using a direct functional approach, involving gating of current measurements and analysis of the Q(V) curves spanning 800 mV in the voltage axis. Systematic neutralization of the individual charged residues in the VSD (S1–S4) revealed that only the neutralization of two arginines in S4 (R210 and R213) changed the voltage dependence of the Q(V) curves. Neutralization of other VSD charges point to roles in tuning of the half-activation voltage of the VSD and its allosteric coupling with the PD. Molecular dynamics (MD) simulations based on the cryo-EM structures of the human BK channel (35) as templates suggested that R210 and R213 lie in a very narrow septum separating intra- and extracellular water-filled vestibules. This interpretation is consistent with the robust hyperpolarization-activated proton currents generated when R210 is mutated to the protonable amino acid histidine. Overall, our results point to a unique and distinctive mode of activation in BK: In contrast to Kv channels, where positive charges move one by one through a charge transfer center (absent in BK channels) that spans the entire electric field (36, 37), charge movement in BK channels is limited to the small displacement of R210 and R213, which itself constitutes a narrow septum where the electric field drops.     

COVID-19 vaccine-induced lymphadenopathies: incidence,course and imaging features from an ultrasound prospective study

Aimslymphadenopathy can occur after COVID-19 vaccination and when encountered at ultrasound examinations performed for other reasons might pose a diagnostic challenge. Purpose of the study was to evaluate the incidence, course and ultrasound imaging features of vaccine-induced lymphadenopathy.Methods89 healthy volunteers (median age 30, 76 females) were prospectively enrolled. Vaccine-related clinical side effects (e.g., fever, fatigue, palpable or painful lymphadenopathy) were recorded. Participants underwent bilateral axillary, supraclavicular and cervical lymph node stations ultrasound 1–4 weeks after the second dose and then again after 4–12 weeks in those who showed lymphadenopathy at the first ultrasound. B-mode, color-Doppler assessment, and shear-wave elastography (SWE) evaluation were performed. The correlation between lymphadenopathy and vaccine-related side effects was assessed using the Fisher’s exact test.ResultsPost-vaccine lymphadenopathy were found in 69/89 (78%) participants (37 single and 32 multiple lymphadenopathy). Among them, 60 presented vaccine-related side effects, but no statistically significant difference was observed between post-vaccine side effect and lymphadenopathy. Ultrasound features of vaccine-related lymphadenopathy consisted of absence of fatty hilum, round shape and diffuse or asymmetric cortical thickness (median cortical thickness of 5 mm). Vascular signal was mainly found to be increased, localized in both central and peripheral regions. SWE showed a soft cortical consistence in all cases (median value 11 Kpa). At follow-up, lymph-node morphology was completely restored in most cases (54/69, 78%) and in no case lymphadenopathy had worsened.ConclusionA high incidence of vaccine-induced lymphadenopathy was found in a population of healthy subjects, with nearly complete regression within 4–12 weeks.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40477-022-00674-3.