Activation of Neurotoxic Astrocytes Due to Mitochondrial Dysfunction Triggered by POLG Mutation.

Mitochondrial diseases are associated with neuronal death and mtDNA depletion. Astrocytes respond to injury or stimuli and damage to the central nervous system. Neurodegeneration can cause astrocytes to activate and acquire toxic functions that induce neuronal death. However, astrocyte activation and its impact on neuronal homeostasis in mitochondrial disease remain to be explored. Using patient cells carrying POLG mutations, we generated iPSCs and then differentiated these into astrocytes. POLG astrocytes exhibited mitochondrial dysfunction including loss of mitochondrial membrane potential, energy failure, loss of complex I and IV, disturbed NAD+/NADH metabolism, and mtDNA depletion. Further, POLG derived astrocytes presented an A1-like reactive phenotype with increased proliferation, invasion, upregulation of pathways involved in response to stimulus, immune system process, cell proliferation and cell killing. Under direct and indirect co-culture with neurons, POLG astrocytes manifested a toxic effect leading to the death of neurons. We demonstrate that mitochondrial dysfunction caused by POLG mutations leads not only to intrinsic defects in energy metabolism affecting both neurons and astrocytes, but also to neurotoxic damage driven by astrocytes. These findings reveal a novel role for dysfunctional astrocytes that contribute to the pathogenesis of POLG diseases.

Hallmark Molecular and Pathological Features of POLG Disease are Recapitulated in Cerebral Organoids.

In this research, a 3D brain organoid model is developed to study POLG-related encephalopathy, a mitochondrial disease stemming from POLG mutations. Induced pluripotent stem cells (iPSCs) derived from patients with these mutations is utilized to generate cortical organoids, which exhibited typical features of the diseases with POLG mutations, such as altered morphology, neuronal loss, and mitochondiral DNA (mtDNA) depletion. Significant dysregulation is also identified in pathways crucial for neuronal development and function, alongside upregulated NOTCH and JAK-STAT signaling pathways. Metformin treatment ameliorated many of these abnormalities, except for the persistent affliction of inhibitory dopamine-glutamate (DA GLU) neurons. This novel model effectively mirrors both the molecular and pathological attributes of diseases with POLG mutations, providing a valuable tool for mechanistic understanding and therapeutic screening for POLG-related disorders and other conditions characterized by compromised neuronal mtDNA maintenance and complex I deficiency.

The NAD+ Precursor Nicotinamide Riboside Rescues Mitochondrial Defects and Neuronal Loss in iPSC derived Cortical Organoid of Alpers' Disease.

Alpers' syndrome is an early-onset neurodegenerative disorder usually caused by biallelic pathogenic variants in the gene encoding the catalytic subunit of polymerase-gamma (POLG), which is essential for mitochondrial DNA (mtDNA) replication. The disease is progressive, incurable, and inevitably it leads to death from drug-resistant status epilepticus. The neurological features of Alpers' syndrome are intractable epilepsy and developmental regression, with no effective treatment; the underlying mechanisms are still elusive, partially due to lack of good experimental models. Here, we generated the patient derived induced pluripotent stem cells (iPSCs) from one Alpers' patient carrying the compound heterozygous mutations of A467T (c.1399G>A) and P589L (c.1766C>T), and further differentiated them into cortical organoids and neural stem cells (NSCs) for mechanistic studies of neural dysfunction in Alpers' syndrome. Patient cortical organoids exhibited a phenotype that faithfully replicated the molecular changes found in patient postmortem brain tissue, as evidenced by cortical neuronal loss and depletion of mtDNA and complex I (CI). Patient NSCs showed mitochondrial dysfunction leading to ROS overproduction and downregulation of the NADH pathway. More importantly, the NAD+ precursor nicotinamide riboside (NR) significantly ameliorated mitochondrial defects in patient brain organoids. Our findings demonstrate that the iPSC model and brain organoids are good in vitro models of Alpers' disease; this first-in-its-kind stem cell platform for Alpers' syndrome enables therapeutic exploration and has identified NR as a viable drug candidate for Alpers' disease and, potentially, other mitochondrial diseases with similar causes.

New drug discovery of cardiac anti-arrhythmic drugs: insights in animal models.

Cardiac rhythm regulated by micro-macroscopic structures of heart. Pacemaker abnormalities or disruptions in electrical conduction, lead to arrhythmic disorders may be benign, typical, threatening, ultimately fatal, occurs in clinical practice, patients on digitalis, anaesthesia or acute myocardial infarction. Both traditional and genetic animal models are: In-vitro: Isolated ventricular Myocytes, Guinea pig papillary muscles, Patch-Clamp Experiments, Porcine Atrial Myocytes, Guinea pig ventricular myocytes, Guinea pig papillary muscle: action potential and refractory period, Langendorff technique, Arrhythmia by acetylcholine or potassium. Acquired arrhythmia disorders: Transverse Aortic Constriction, Myocardial Ischemia, Complete Heart Block and AV Node Ablation, Chronic Tachypacing, Inflammation, Metabolic and Drug-Induced Arrhythmia. In-Vivo: Chemically induced arrhythmia: Aconitine antagonism, Digoxin-induced arrhythmia, Strophanthin/ouabain-induced arrhythmia, Adrenaline-induced arrhythmia, and Calcium-induced arrhythmia. Electrically induced arrhythmia: Ventricular fibrillation electrical threshold, Arrhythmia through programmed electrical stimulation, sudden coronary death in dogs, Exercise ventricular fibrillation. Genetic Arrhythmia: Channelopathies, Calcium Release Deficiency Syndrome, Long QT Syndrome, Short QT Syndrome, Brugada Syndrome. Genetic with Structural Heart Disease: Arrhythmogenic Right Ventricular Cardiomyopathy/Dysplasia, Dilated Cardiomyopathy, Hypertrophic Cardiomyopathy, Atrial Fibrillation, Sick Sinus Syndrome, Atrioventricular Block, Preexcitation Syndrome. Arrhythmia in Pluripotent Stem Cell Cardiomyocytes. Conclusion: Both traditional and genetic, experimental models of cardiac arrhythmias' characteristics and significance help in development of new antiarrhythmic drugs.

Application of Flow Cytometric Analysis for Measuring Multiple Mitochondrial Parameters in 3D Brain Organoids.

Mitochondrial dysfunction is a common primary or secondary contributor to many types of neurodegeneration, and changes in mitochondrial mass, mitochondrial respiratory chain (MRC) complexes, and mitochondrial DNA (mtDNA) copy number often feature in these processes. Human brain organoids derived from human induced pluripotent stem cells (iPSCs) recapitulate the brain's three-dimensional (3D) cytoarchitectural arrangement and offer the possibility to study disease mechanisms and screen new therapeutics in a complex human system. Here, we report a unique flow cytometry-based approach to measure multiple mitochondrial parameters in iPSC-derived cortical organoids. This report details a protocol for generating cortical brain organoids from iPSCs, single-cell dissociation of generated organoids, fixation, staining, and subsequent flow cytometric analysis to assess multiple mitochondrial parameters. Double staining with antibodies against the MRC complex subunit NADH: Ubiquinone Oxidoreductase Subunit B10 (NDUFB10) or mitochondrial transcription factor A (TFAM) together with voltage-dependent anion-selective channel 1 (VDAC 1) permits assessment of the amount of these proteins per mitochondrion. Since the quantity of TFAM corresponds to the amount of mtDNA, it provides an indirect estimation of the number of mtDNA copies per mitochondrial content. This entire procedure can be completed within a span of 2-3 h. Crucially, it allows for the concurrent quantification of multiple mitochondrial parameters, including both total and specific levels relative to the mitochondrial mass.

Antimicrobial Susceptibility Trends among Pathogens Isolated from Blood: A 6-Year Retrospective Study from a Tertiary Care Hospital in East Sikkim, India.

Background Bloodstream infections (BSIs) are one of the frequent nosocomial infections among hospitalized patients. To understand the local epidemiology and evolving antimicrobial drug resistance of blood-borne pathogens, we analyzed the distribution and antibiotic sensitivity profile of organisms causing BSI in our hospital-based study. Materials and Methods We reviewed retrospective data of laboratory-confirmed BSIs, from January 2013 to December 2018. Causative organisms and their antibiotic susceptibility profile of primary and secondary BSI reports were determined from BacT/Alert and Vitek systems findings (bioMérieux). A 6-year multidrug resistance indexing was done to document the resistance pattern of the commonly isolated organisms. Results A total of 1,340 (10.2%) BSIs were reported from 13,091 blood cultures. Organisms were frequently isolated from the younger population (≤20 years), especially from ages < 1 year (20.8% of total BSIs). Majority of pathogens were bacterial (97.1%) whereas 2.9% were fungal in origin. Monomicrobial growth was recorded in over 98% of BSIs. Gram-positive and gram-negative bacteria isolated were 518 (39.8%) and 783 (60.2%), respectively. Commonly isolated organisms were coagulase-negative Staphylococci (29.4%), Escherichia coli (19.8%), Klebsiella species (13.5%), Salmonella species (9.4%), and Staphylococcus aureus (7.5%). Multidrug-resistance index was observed highest in Acinetobacter species followed by Pseudomonas aeruginosa and S. aureus . Conclusion Overall, there has been a gradual decline in the reporting of BSI. However, infections by gram-negative bacilli and multidrug-resistant organisms remain persistently high. Ages < 20 years were the vulnerable group, with infants < 1 year contributing to the maximum number of BSI cases caused by both bacteria and fungi. Therefore, additional methods are required to study the origin and causation of these infections, particularly among vulnerable patients.

Study of vancomycin and high-level aminoglycoside-resistant Enterococcus species and evaluation of a rapid spot test for enterococci from Central Referral Hospital, Sikkim, India.

BACKGROUND: Enterococcus is an important pathogen, and with its emergence of resistance to multiple antimicrobials, the management of infection is becoming increasingly difficult. AIM: The aim of the study is to determine the prevalence, antibiotic resistance, and risk factors associated with enterococcal infection or colonization. MATERIALS AND METHODS: In this prospective study, samples from inpatients were screened for resistant enterococci. Antibiotic susceptibility testing was performed using the disc diffusion method and minimum inhibitory concentration by the agar dilution method. A modification of a test tube method of sodium chloride-esculin hydrolysis to a spot test was evaluated for its rapidity and reliability in the presumptive diagnosis of enterococci. STATISTICAL ANALYSIS USED: Fisher's exact test was used for continuous (Student's t-test) and categorical variables. Multivariate analysis was performed with logistic regression using IBM SPSS 20.0 software (Armonk, NY, USA). RESULTS: Enterococcus species were isolated from 182 samples: Enterococcus faecalis (68.7%), Enterococcus faecium (20.9%), Enterococcus gallinarum (6%), and Enterococcus durans (4.4%). Maximum resistance was to ciprofloxacin (59.3%) and least to linezolid (0.5%). The isolation rate of vancomycin-resistant enterococci (VRE) was 13.7%; 30.2% and 20.9% were of high-level gentamicin and streptomycin, respectively. All 182 Enterococcus species gave positive results within 30-60 min by the rapid spot test. CONCLUSIONS: Overall, high-level aminoglycoside resistance (HLAR) was observed more than glycopeptide resistance. Surveillance strategies need to be upgraded and implemented in order to prevent the emergence and further spread of not only VRE but also HLAR enterococci in the hospital. The spot test gave reliable and rapid results in presumptive identification of enterococci.