Gradual ER calcium depletion induces a progressive and reversible UPR signaling.

The unfolded protein response (UPR) is a widespread signal transduction pathway triggered by endoplasmic reticulum (ER) stress. Because calcium (Ca2+) is a key factor in the maintenance of ER homeostasis, massive Ca2+ depletion of the ER is a potent inducer of ER stress. Although moderate changes in ER Ca2+ drive the ubiquitous Ca2+ signaling pathways, a possible incremental relationship between UPR activation and Ca2+ changes has yet to be described. Here, we determine the sensitivity and time-dependency of activation of the three ER stress sensors, inositol-requiring protein 1 alpha (IRE1α), protein kinase R-like ER kinase (PERK), and activating transcription factor 6 alpha (ATF6α) in response to controlled changes in the concentration of ER Ca2+ in human cultured cells. Combining Ca2+ imaging, fluorescence recovery after photobleaching experiments, biochemical analyses, and mathematical modeling, we uncover a nonlinear rate of activation of the IRE1α branch of UPR, as compared to the PERK and ATF6α branches that become activated gradually with time and are sensitive to more important ER Ca2+ depletions. However, the three arms are all activated within a 1 h timescale. The model predicted the deactivation of PERK and IRE1α upon refilling the ER with Ca2+. Accordingly, we showed that ER Ca2+ replenishment leads to the complete reversion of IRE1α and PERK phosphorylation in less than 15 min, thus revealing the highly plastic character of the activation of the upstream UPR sensors. In conclusion, our results reveal a dynamic and dose-sensitive Ca2+-dependent activation/deactivation cycle of UPR induction, which could tightly control cell fate upon acute and/or chronic stress.

T cell Ca2+ microdomains through the lens of computational modeling.

Cellular Ca2+ signaling is highly organized in time and space. Locally restricted and short-lived regions of Ca2+ increase, called Ca2+ microdomains, constitute building blocks that are differentially arranged to create cellular Ca2+ signatures controlling physiological responses. Here, we focus on Ca2+ microdomains occurring in restricted cytosolic spaces between the plasma membrane and the endoplasmic reticulum, called endoplasmic reticulum-plasma membrane junctions. In T cells, these microdomains have been finely characterized. Enough quantitative data are thus available to develop detailed computational models of junctional Ca2+ dynamics. Simulations are able to predict the characteristics of Ca2+ increases at the level of single channels and in junctions of different spatial configurations, in response to various signaling molecules. Thanks to the synergy between experimental observations and computational modeling, a unified description of the molecular mechanisms that create Ca2+ microdomains in the first seconds of T cell stimulation is emerging.

Computational investigation of IP3 diffusion.

Inositol 1,4,5-trisphosphate (IP3) plays a key role in calcium signaling. After stimulation, it diffuses from the plasma membrane where it is produced to the endoplasmic reticulum where its receptors are localized. Based on in vitro measurements, IP3 was long thought to be a global messenger characterized by a diffusion coefficient of ~ 280 µm2s-1. However, in vivo observations revealed that this value does not match with the timing of localized Ca2+ increases induced by the confined release of a non-metabolizable IP3 analog. A theoretical analysis of these data concluded that in intact cells diffusion of IP3 is strongly hindered, leading to a 30-fold reduction of the diffusion coefficient. Here, we performed a new computational analysis of the same observations using a stochastic model of Ca2+ puffs. Our simulations concluded that the value of the effective IP3 diffusion coefficient is close to 100 µm2s-1. Such moderate reduction with respect to in vitro estimations quantitatively agrees with a buffering effect by non-fully bound inactive IP3 receptors. The model also reveals that IP3 spreading is not much affected by the endoplasmic reticulum, which represents an obstacle to the free displacement of molecules, but can be significantly increased in cells displaying elongated, 1-dimensional like geometries.

A journey in UPR modelling.

Protein folding and protein maturation largely occur in the controlled environment of the Endoplasmic Reticulum (ER). Perturbation to the correct functioning of this organelle leads to altered proteostasis and accumulation of misfolded proteins in the ER lumen. This condition is commonly known as ER stress and is appearing as an important contributor in the pathogenesis of several human diseases. Monitoring of the quality control processes is mediated by the Unfolded Protein Response (UPR). This response consists in a complex network of signalling pathways that aim to restore protein folding and ER homeostasis. Conditions in which UPR is not able to overcome ER stress lead to a switch of the UPR signalling program from an adaptive to a pro-apoptotic one, revealing a key role of UPR in modulating cell fate decisions. Because of its high complexity and its involvement in the regulation of different cellular outcomes, UPR has been the centre of the development of computational models, which tried to better dissect the role of UPR or of its specific components in several contexts. In this review, we go through the existing mathematical models of UPR. We emphasize how their study contributed to an improved characterization of the role of this intricate response in the modulation of cellular functions.

Efficacy of VERIGENE® Blood Culture Assays in Accurately and Rapidly Detecting Gram-Negative and Gram-Positive Pathogens.

BACKGROUND: Rapid administration of appropriately indicated antibiotics is crucial in septic patients. Sepsis data supports that there is a higher risk of mortality for each hour delay from triage to antibiotic therapy, as well as for inappropriate antibiotic selection. There are a variety of rapid microbial detection systems, such as VERIGENE®, used in acute care facilities to rapidly detect bacteremia and identify resistance markers. Our study investigates the usefulness of VERIGENE® assays in accurately detecting Gram-positive and Gram-negative pathogens when compared to traditional blood culture analysis systems, such as VITEK®. METHODS: 819 Gram-positive and 373 Gram-negative blood samples were collected and tested using both VERIGENE® and VITEK®. Statistical tests were two-tailed and observations were defined as statistically significant if P ≤ 0.05. RESULTS: VERIGENE® detected a pathogen in 816/819 (99.6%) samples of the Gram-positive blood cultures and 367/373 (98.3%) samples of the Gram-negatives compared to 805/819 (98.3%) and 367/373 (98.4%), respectively, using VITEK®. Gram-positive cultures had a sensitivity of 99.5% and a specificity of 27.3% (PPV 99.0%, NPV 42.9%, 98.7% accuracy) with VERIGENE analysis. Gramnegatives had a sensitivity of 99.2% and a specificity of 20.0% (PPV 98.9%, NPV 25.0%, 98.4% accuracy). CONCLUSION: Although statistically insignificant (P = 0.25), VERIGENE® was 1.3% more likely to identify Gram-positive bacteria when compared to conventional methods. Overall, we concluded that VERIGENE® assays are valuable in their ability to rapidly detect microorganisms and resistance markers, given their high sensitivities. This allows for select targeted therapy in patients with sepsis and can ultimately reduce mortality rates.

The Use of Single Therapy With Tocilizumab Versus Combination Therapy With Remdesivir and Tocilizumab in SARS-CoV-2 Patients in El Paso, Texas.

BACKGROUND: Currently, the management of SARS-CoV-2 varies with no definitive clinical guidelines, as scientific evidence across the globe differs in therapeutic options. This study intended to provide some clarity to the insufficient data based on the role of monotherapy with tocilizumab (TCZ) and combination therapy with remdesivir (RDV) and TCZ among patients with SARS-CoV-2 infection in El Paso, Texas. We evaluated the use of each therapy in the presence of steroids as the standard of care. METHODS:  One hundred and fifty-four SARS-CoV-2-infected patients from four different medical centers in El Paso, Texas, were screened, with 113 eligible for this longitudinal comparative observational study (February 1, 2020 to October 31, 2020). Group 1 (80 patients) received TCZ in the first 24 hours following admission, then methylprednisolone for the next 72 hours and group 2 (33 patients) were given TCZ as detailed in the single therapy group, plus RDV within the first 24 hours. Mann Whitney U test assessed median differences in laboratory biomarkers and Bivariate Logistic Regression assessed the odds of risk. An observation is considered statistically significant when P-value is ≤0.05. RESULTS: Patients in group 1 had a statistically significant lower odds for ventilation use than group 2 (OR=0.34, 95%CI=0.12-0.95, p=0.034), although no statistically significant difference in mortality outcomes was observed across groups (OR=0.43, 95%CI:0.13-1.39, p=0.269). CONCLUSIONS: We concluded that the use of TCZ in SARS-CoV-2-infected patients in El Paso, with or without RDV, reported no mortality benefit. However, some minimal/non-use of ventilation benefit was observed in group 1. Nonetheless, a randomized controlled trial study is recommended to ultimately determine the combination role of TCZ and RDV among this highly vulnerable group of patients.

Risk factors for mortality among patients with SARS-CoV-2 infection: A longitudinal observational study.

Recent literature suggests that approximately 5%-18% of patients diagnosed with severe acute respiratory syndrome coronavirus 2 may progress rapidly to a severe form of the illness and subsequent death. We examined the relationship between sociodemographic, clinical, and laboratory findings with mortality among patients. In this study, 112 patients were evaluated from February to May 2020 and 80 patients met the inclusion criteria. Tocilizumab was administered, followed by methylprednisolone to patients with pneumonia severity index score ≤130 and computerized tomography scan changes. Demographic data and clinical outcomes were collected. Laboratory biomarkers were monitored during hospitalization. Statistical analyses were performed with significance p ≤ .05. A total of 80 patients: 45 males (56.25%) and 35 females (43.75%) met the study inclusion criteria. A total of 7 patients (8.75%) were deceased. An increase in mortality outcome was statistically significantly associated with higher average levels of interleukin-6 (IL-6) with p value (.050), and d-dimer with p value (.024). Bivariate logistics regression demonstrated a significant increased odds for mortality for patients with bacterial lung infections (odds ratio [OR]: 10.83; 95% confidence interval [CI]: 2.05-57.40; p = .005) and multiorgan damage (OR: 103.50; 95% CI: 9.92-1079.55; p = .001). Multivariate logistics regression showed a statistically significant association for multiorgan damage (adjusted odds ratio [AOR]: 94.17; 95% CI: 7.39-1200.78; p = .001). We identified three main predictors for high mortality. These include IL-6, d-dimer, and multiorgan damage. The latter was the highest potential risk for in-hospital deaths. This warrants aggressive health measures for early recognition of the problem and initiation of treatment to reverse injuries.

Early use of tocilizumab in the prevention of adult respiratory failure in SARS-CoV-2 infections and the utilization of interleukin-6 levels in the management.

Respiratory failure in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection appears related to cytokine release syndrome that often results in mechanical ventilation (MV). We investigated the role of tocilizumab (TCZ) on interleukin-6 (IL-6) trends and MV in patients with SARS-CoV-2. In this longitudinal observational study, 112 patients were evaluated from 1 February to 31 May 2020. TCZ was administered followed by methylprednisolone to patients with >3L oxygen requirement and pneumonia severity index score ≤130 with computed tomography scan changes. IL-6, C-reactive protein (CRP), ferritin, lactate dehydrogenase (LDH), D-dimer, and procalcitonin were monitored on days 0, 3, and 6 of therapy. Statistical analyses were performed with significance ≤0.05. Eighty out of 112 SARS-CoV-2-positive patients (45 males, 56.96%; 34 females, 43.04%) were included in this study. Seven patients expired (8.75%) and nine patients required MV (11.25%). Median IL-6 levels pre-administration of TCZ was 342.50 (78.25-666.25) pg/mL compared with post-administration on day 3 (563; 162-783) pg/mL (P < .00001). On day 6, the median dropped to 545 (333.50-678.50) pg/mL compared with day 3 (P = .709). CRP, ferritin, LDH, and D-dimer levels were reduced after TCZ therapy. Early use of TCZ may reduce the need for MV and decrease CRP, ferritin, LDH, and D-dimer levels. The sequential use of methylprednisolone for 72 hours seems to potentiate the effect and prolong the suppression of the cytokine storm. IL-6 levels may be helpful as a prognostic tool.

Orofacial motor dysfunction in Moebius syndrome.

AIM: To review orofacial disabilities and their consequences in children with Moebius syndrome (MBS). METHOD: We retrospectively analysed the records of 32 patients (21 males, 11 females) with non-progressive bilateral facial and abducens palsies who had been examined before 6 months of age. RESULTS: All facial muscles were severely involved in 17 patients; in the 15 others, partial movements were found in the lower face. Most patients (n=24) were unable to smile. Patients frequently presented with congenital trismus (n=20) and drooling (n=18). Additional palsies involved cranial nerves IX and X (n=18) and XII (n=25). Sucking was absent or weak in 30 patients; swallowing was impaired in 25. During the first month of life, feeding disorders were graded as severe/moderate in 25. Respiratory complications occurred in 17. Severe feeding disorders were associated with congenital trismus (p=0.01) and with cranial nerve IX and X palsy (p=0.01). Growth failure between 1 and 6 months of age, followed by catch-up growth between 6 and 12 months, was observed in 20 patients. Between 2 and 5 years of age, 25 out of 32 patients attained normal oral diet and 28 out of 29 showed normal growth. INTERPRETATION: Children with MBS frequently require adjusted therapeutic options to prevent failure to thrive. Congenital trismus, cranial nerve IX and X palsy, and laryngeal-tracheal dysfunctions are predictors of severe feeding disorders. WHAT THIS PAPER ADDS: Moebius syndrome frequently induces reduced oral intake and early failure to thrive. Normal oral diet and growth parameters are attained at 2 to 5 years of age. Congenital trismus, pharyngeal palsy, and laryngeal disorders predict dysphagia.

Pathogenesis of cranial neuropathies in Moebius syndrome: Electrodiagnostic orofacial studies.

INTRODUCTION: We designed a retrospective study of 59 patients with congenital sporadic nonprogressive bilateral facial and abducens palsies. METHODS: Examinations included needle electromyography (EMG) of facial and oral muscles, facial nerve motor latency and conduction velocity (FNCV), and blink responses (BR). RESULTS: Neurogenic EMG changes were found in 1 or more muscles in 55 of 59 patients, with no abnormal spontaneous activity. EMG changes were homogeneously neurogenic in 17 patients, homogeneously myopathic in 1 patient, and heterogeneous in 41 of 59 patients. Motor latency was increased according to recordings from 52 of 137 facial muscles. An increase of motor latency was not associated with neurogenic EMG (Fischer's test: right, P = 1; left, P = 0.76). FNCV was slowed in 19 of 36 patients. BR was absent bilaterally in 35 of 58 patients; when present, R1 and R2 latencies were normal. DISCUSSION: Our results support the hypothesis of an early developmental defect localized in motor cranial nerves with spared V-VII internuclear pathways. Muscle Nerve, 2018.

New aspects in the pathomechanism of diseases of civilization, particularly psychosomatic disorders. Part 1. Theoretical background of a hypothesis.

The stress defence-cascade is mostly not biphasic as Cannon thought, the sympathicotonic stress response is preceded by a vagotonic phase called freeze response. Alteration of the carbon dioxide level plays an important role during defence-cascade as its changes interfere with stress hormones, e.g. with catecholamines, thus affecting the degree of arousal. In case of humans, learned behaviour dominates instead of instinctive, so the fight-or-flight often lags; the consequence can be persistent hypocapnia or hypercapnia. The hypoventilation or hyperventilation may continue even after a stressful situation, as tissular and renal compensation stabilizes and makes the pathological breathing patterns chronic. The organism tries to restore the original milieu intérieur (sec. Claude Bernard), but this cannot succeed without restoring pCO2. The regulation operates the preservation of intracellular and extracellular pH as a priority, while neurohumoral compensations change the ionic milieu in the body's cells. Present hypothesis specifies the permanent lack or excess of carbon dioxide which can cause allostatic load by psychosomatic pathomechanism. Carbon dioxide is equivalent to stress hormones; its alterations become a means of somatization, resulting in ion-pattern changes in intracellular and extracellular spaces, consequently causing disintegration of the body's function. (See also: network theory, ripple effect, metabolic remodeling.) Intracellular ion-pattern alterations emerge new genetic phenotypes to the surface. The variety of phenotypes explains the diversity of induced diseases. The theory appreciates the role of ions by considering the instantaneous ion pattern of the cytoplasm (all the ions together) as a decisive second messenger.

New aspects in the pathomechanism of diseases of civilization, particularly psychosomatic disorders. Part 2. Chronic hypocapnia and hypercapnia in the medical practice.

The authors seek to find new connections between recent results of biology and older theories. This paper aims to assemble the jigsaw puzzle. The theoretical background of the hypothesis was described in the previous issue of the journal (Sikter et al. 2017a). Human stress response often coexists with persistent hypocapnia or hypercapnia - developing via psychosomatic pathomechanism - which can lead to mental and psychosomatic illnesses. Chronic hypocapnia mainly generates hyperarousal disorders which may be reversible for an extended time, however, vicious cycles may start when hypoxia and/or severe somatic diseases are simultaneously present (commonly in the elderly), which conditions often end with death without medical help. Chronic hypercapnia devastates the organism initially without symptoms, partly due to neurohumoral contraregulation, consequential dysregulation and metabolic remodeling. Psychosomatic disorders (e.g., diseases of civilization that evolve in people with disadvantaged psychosocial situations) develop over years and decades, causing irreversible changes. Hypercapnia usually occurs in clinical pictures of chronic obstructive pulmonary disease, obesity hypoventilation syndrome, obstructive sleep apnea, and its unobstructed version (sleep-related hypoventilation), generating various organic disorders (hypertension, type 2 diabetes, cardiovascular disorders, immunological diseases, depression, etc.). Because of the above, chronic hypocapnia and hypercapnia cannot be regarded as harmless accompanying phenomena. That is why we have to strive for restoring eucapnia and normalizing the induced ionic changes, which does not appear to be a hopeless task.

Long-term EEG in patients with the ring chromosome 20 epilepsy syndrome.

The recognizable electroencephalography (EEG) pattern of ring chromosome 20 epilepsy syndrome can be missing in patients with r(20) chromosomal anomaly, and may be found in patients with frontal lobe epilepsy of other origin. This study aims to search for more specific EEG signs by using long-term recordings and measuring the duration of paroxysmal anomalies. The series included 12 adult patients with r(20) anomaly, and 12 controls without any chromosomal aberration. We measured the duration of every paroxysmal burst and calculated the sum of their durations for each long-term EEG recording. We compared patients to controls using the Mann-Whitney U-test. Every patient showed long-lasting paroxysmal EEG bursts, up to 60 min; controls did not show any bursts longer than 60 s (p < 0.0001). The total duration of paroxysmal anomalies was significantly longer in patients (31-692 min) compared to controls (0-48 min) (p < 0.0001). Thus, long-term recordings enhance the contribution of EEG methods for characterizing the ring 20 chromosome epilepsy syndrome.

Outcomes of Neonatal Bulbar Weakness.

BACKGROUND AND OBJECTIVE: Neonatal bulbar weakness (BW) has various etiologies and a broad prognostic range. We aimed to report outcomes in a large series of children with neonatal BW and explore the association of orofacial electrodiagnostic data with outcome. METHODS: We retrospectively reviewed the files of children who presented with facial, lingual, laryngeal, or pharyngeal weakness at birth and who underwent electrodiagnostic studies combining conventional needle electromyography (EMG) of orofacial muscles, blink responses, and EMG during bottle-feeding. Outcome measures included the need for prolonged respiratory assistance and enteral feeding, as well as sensorimotor and cognitive impairments. RESULTS: Of 175 patients, 73% had developmental disorders, 25% suffered from acquired brain damage, and 2% had no apparent underlying disorders. Motor or mental impairment was observed in 71%; death occurred in 16%. Outcomes were not significantly different when comparing developmental disorders versus acquired brain damage or neurogenic versus normal detection EMG. Abnormal blink responses were associated with higher frequencies of respiratory assistance (P = .03), gastrostomy (P = .025), and death (P = .009); moderate or severe oropharyngeal incoordinations were associated with higher frequencies of respiratory assistance (P = .006), prolonged enteral feeding (P < .0001), and gastrostomy (P = .0002). CONCLUSIONS: Orofacial electrodiagnostic studies provide supplementary information to help the pediatrician anticipate the management and prognosis of young infants with BW.

Congenital multiple cranial neuropathies: Relevance of orofacial electromyography in infants.

INTRODUCTION: The aim of this study was to assess diagnoses and outcomes of infants with 2 or more cranial neuropathies identified using orofacial electromyography (EMG). METHODS: This retrospective study involved 90 patients. Diagnoses took into account clinical, radiological, and genetic data. EMG examined the orbicularis oculi, genioglossus, and levator veli palatini muscles, and blink responses. To evaluate outcome, neurological disability, respiratory complications, and feeding difficulties were recorded. RESULTS: The patients had malformation syndromes (59), encephalopathies (29), or no underlying disorders (2). Neurogenic EMG signs were detected in a mean of 4 muscles, reflecting a mean of 3 affected nerves. EMG identified a higher number of neuropathies than clinical examination alone (82 vs. 31, facial; 56 vs. 2, pharyngeal; 25 vs. 3, hypoglossal). Poor outcome and death were more frequent when EMG identified ≥4 affected nerves (P = 0.02). CONCLUSION: EMG highlights multiple cranial neuropathies that can be clinically silent in infants with malformation syndromes or encephalopathies.

Facial, lingual, and pharyngeal electromyography in infants with Pierre Robin sequence.

INTRODUCTION: We evaluated the role of electromyography (EMG) in assessing orofacial neurological dysfunction in 81 infants with Pierre Robin sequence (PRS). METHODS: Needle EMG of muscles of the face, tongue, and soft palate, and blink responses were recorded. A two-channel EMG recorded sucking and swallowing during bottle feeding. RESULTS: Neurogenic EMG signs were detected in facial or oral muscles in 17 of 24 associated PRS and 1 of 57 isolated PRS cases (P < 0.0001). Soft palate muscles showed low-amplitude traces in 41.4% of patients who required two surgical steps for cleft palate repair and 18.5% of those who required only one step. Regarding EMG study during bottle feeding, patients with moderate or severe abnormalities of oral/pharyngeal coordination required more prolonged enteral feeding than patients with mild abnormalities or normal coordination (P = 0.002). CONCLUSION: Combined EMG methods were useful in the treatment of infants with PRS. EMG detection of cranial nerve involvement strongly suggests an associated form of PRS.

Usher syndrome type 1: early detection of electroretinographic changes.

BACKGROUND: Usher syndrome type 1 needs to be diagnosed at early age in order to timely manage speech therapy, cochlear implantation, and genetic counseling. Few data are available regarding electroretinographic testing before the age of six years. AIM: To describe electroretinographic changes in young children with Usher syndrome type 1. METHODS: Retrospective study of fourteen patients. Age at first neurophysiologic testing was between 17 months and 5 years 4 months. Electroretinogram was performed using flash stimulation in mesopic conditions in the conscious child. Analysis was focused on the amplitudes and latencies of a- and b-waves. RESULTS: Whatever the age, an abnormal fundus was always confirmed with an absent electroretinogram. The youngest patient with absent electroretinogram was 17 month-old. When recorded on and after the 29th month of age, electroretinogram was absent in all cases, including 6 patients with normal fundus. In three patients a low-amplitude electroretinogram was present at first recording within the 26th and 27th months. CONCLUSION: Electroretinogram showed retinopathy in young children with Usher syndrome type 1, even in the absence of fundoscopic signs of retinal degeneration.

Positive slow waves in the EEG of premature infants between 24 and 36 weeks of conceptional age.

OBJECTIVE: The aim of this prospective and longitudinal study was to characterize EEG patterns during the first weeks of life in extremely premature infants. METHODS: Twenty-five extremely premature infants were included and weekly EEG recordings were obtained between 24 and 36 weeks of conceptional age (CA). RESULTS: Central (rolandic) positive slow waves (CPSW) were found to be the most reliable and characteristic pattern. CPSWs were frequent at 24 weeks CA and progressively diminished and disappeared around 34 weeks CA. CPSWs appeared isolated or in sequences, they occurred during periods of continuous or discontinuous EEG activity, during bursts or during intervals of discontinuous activity, and unilaterally or bilaterally. Temporal positive slow waves and theta rhythms occurred less often and did not decrease as a function of CA. In addition, the amount of discontinuity progressively decreased until 36 weeks CA. The duration of bursts stayed constant, while inter-burst intervals decreased as a function of CA. CONCLUSIONS: Central positive slow waves are characteristic features of the EEG in the extremely premature infant. SIGNIFICANCE: The presence of CPSWs and their progressive disappearance until 34 weeks CA may represent a maturational marker in the EEG.

Cytogenetic analysis of the mononuclear cell component of giant cell tumors of bone.

This study comprises the cytogenetic analysis of mononuclear cells of a Mexican patient with a giant cell tumor of bone. This cell line showed nine translocations, two duplications, one addition, one deletion, and one ring chromosome, and did not present telomeric association.