Mitochondrial DNA competition: starving out the mutant genome.

High levels of pathogenic mitochondrial DNA (mtDNA) variants lead to severe genetic diseases, and the accumulation of such mutants may also contribute to common disorders. Thus, selecting against these mutants is a major goal in mitochondrial medicine. Although mutant mtDNA can drift randomly, mounting evidence indicates that active forces play a role in the selection for and against mtDNA variants. The underlying mechanisms are beginning to be clarified, and recent studies suggest that metabolic cues, including fuel availability, contribute to shaping mtDNA heteroplasmy. In the context of pathological mtDNAs, remodeling of nutrient metabolism supports mitochondria with deleterious mtDNAs and enables them to outcompete functional variants owing to a replicative advantage. The elevated nutrient requirement represents a mutant Achilles' heel because small molecules that restrict nutrient consumption or interfere with nutrient sensing can purge cells of deleterious mtDNAs and restore mitochondrial respiration. These advances herald the dawn of a new era of small-molecule therapies to counteract pathological mtDNAs.

Two mitochondrial DNA polymorphisms modulate cardiolipin binding and lead to synthetic lethality.

Genetic screens have been used extensively to probe interactions between nuclear genes and their impact on phenotypes. Probing interactions between mitochondrial genes and their phenotypic outcome, however, has not been possible due to a lack of tools to map the responsible polymorphisms. Here, using a toolkit we previously established in Drosophila, we isolate over 300 recombinant mitochondrial genomes and map a naturally occurring polymorphism at the cytochrome c oxidase III residue 109 (CoIII109) that fully rescues the lethality and other defects associated with a point mutation in cytochrome c oxidase I (CoIT300I). Through lipidomics profiling, biochemical assays and phenotypic analyses, we show that the CoIII109 polymorphism modulates cardiolipin binding to prevent complex IV instability caused by the CoIT300I mutation. This study demonstrates the feasibility of genetic interaction screens in animal mitochondrial DNA. It unwraps the complex intra-genomic interplays underlying disorders linked to mitochondrial DNA and how they influence disease expression.

Synchronization of spin-driven limit cycle oscillators optically levitated in vacuum.

We explore, experimentally and theoretically, the emergence of coherent coupled oscillations and synchronization between a pair of non-Hermitian, stochastic, opto-mechanical oscillators, levitated in vacuum. Each oscillator consists of a polystyrene microsphere trapped in a circularly polarized, counter-propagating Gaussian laser beam. Non-conservative, azimuthal forces, deriving from inhomogeneous optical spin, push the micro-particles out of thermodynamic equilibrium. For modest optical powers each particle shows a tendency towards orbital circulation. Initially, their stochastic motion is weakly correlated. As the power is increased, the tendency towards orbital circulation strengthens and the motion of the particles becomes highly correlated. Eventually, centripetal forces overcome optical gradient forces and the oscillators undergo a collective Hopf bifurcation. For laser powers exceeding this threshold, a pair of limit cycles appear, which synchronize due to weak optical and hydrodynamic interactions. In principle, arrays of such Non-Hermitian elements can be arranged, paving the way for opto-mechanical topological materials or, possibly, classical time crystals. In addition, the preparation of synchronized states in levitated optomechanics could lead to new and robust sensors or alternative routes to the entanglement of macroscopic objects.

Milton assembles large mitochondrial clusters, mitoballs, to sustain spermatogenesis.

Mitochondria are dynamic organelles that undergo frequent remodeling to accommodate developmental needs. Here, we describe a striking organization of mitochondria into a large ball-like structure adjacent to the nucleus in premeiotic Drosophila melanogaster spermatocytes, which we term "mitoball". Mitoballs are transient structures that colocalize with the endoplasmic reticulum, Golgi bodies, and the fusome. We observed similar premeiotic mitochondrial clusters in a wide range of insect species, including mosquitos and cockroaches. Through a genetic screen, we identified that Milton, an adaptor protein that links mitochondria to microtubule-based motors, mediates mitoball formation. Flies lacking a 54 amino acid region in the C terminus of Milton completely lacked mitoballs, had swollen mitochondria in their spermatocytes, and showed reduced male fertility. We suggest that the premeiotic mitochondrial clustering is a conserved feature of insect spermatogenesis that supports sperm development.

Rapid Identification of Pathogens Causing Bloodstream Infections by Raman Spectroscopy and Raman Tweezers.

The search for the "Holy Grail" in clinical diagnostic microbiology-a reliable, accurate, low-cost, real-time, easy-to-use method-has brought up several methods with the potential to meet these criteria. One is Raman spectroscopy, an optical, nondestructive method based on the inelastic scattering of monochromatic light. The current study focuses on the possible use of Raman spectroscopy for identifying microbes causing severe, often life-threatening bloodstream infections. We included 305 microbial strains of 28 species acting as causative agents of bloodstream infections. Raman spectroscopy identified the strains from grown colonies, with 2.8% and 7% incorrectly identified strains using the support vector machine algorithm based on centered and uncentred principal-component analyses, respectively. We combined Raman spectroscopy with optical tweezers to speed up the process and captured and analyzed microbes directly from spiked human serum. The pilot study suggests that it is possible to capture individual microbial cells from human serum and characterize them by Raman spectroscopy with notable differences among different species. IMPORTANCE Bloodstream infections are among the most common causes of hospitalizations and are often life-threatening. To establish an effective therapy for a patient, the timely identification of the causative agent and characterization of its antimicrobial susceptibility and resistance profiles are essential. Therefore, our multidisciplinary team of microbiologists and physicists presents a method that reliably, rapidly, and inexpensively identifies pathogens causing bloodstream infections-Raman spectroscopy. We believe that it might become a valuable diagnostic tool in the future. Combined with optical trapping, it offers a new approach where the microorganisms are individually trapped in a noncontact way by optical tweezers and investigated by Raman spectroscopy directly in a liquid sample. Together with the automatic processing of measured Raman spectra and comparison with a database of microorganisms, it makes the whole identification process almost real time.

SERS-Tags: Selective Immobilization and Detection of Bacteria by Strain-Specific Antibodies and Surface-Enhanced Raman Scattering.

Efficient separation and sensitive identification of pathogenic bacterial strains is essential for a prosperous modern society, with direct applications in medical diagnostics, drug discovery, biodefense, and food safety. We developed a fast and reliable method for antibody-based selective immobilization of bacteria from suspension onto a gold-plated glass surface, followed by detection using strain-specific antibodies linked to gold nanoparticles decorated with a reporter molecule. The reporter molecules are subsequently detected by surface-enhanced Raman spectroscopy (SERS). Such a multi-functionalized nanoparticle is called a SERS-tag. The presented procedure uses widely accessible and cheap materials for manufacturing and functionalization of the nanoparticles and the immobilization surfaces. Here, we exemplify the use of the produced SERS-tags for sensitive single-cell detection of opportunistic pathogen Escherichia coli, and we demonstrate the selectivity of our method using two other bacterial strains, Staphylococcus aureus and Serratia marcescens, as negative controls. We believe that the described approach has a potential to inspire the development of novel medical diagnostic tools for rapid identification of bacterial pathogens.

Pharmacokinetics and Pharmacodynamics of a Novel U500 Insulin Aspart Formulation: A Randomized, Double-Blind, Crossover Study in People With Type 1 Diabetes.

OBJECTIVE: To evaluate the pharmacokinetics, pharmacodynamics, and safety of a novel U500 insulin aspart formulation (AT278 U500) compared with insulin aspart (IAsp U100). RESEARCH DESIGN AND METHODS: This single-center, randomized, double-blind study was conducted in 38 men with type 1 diabetes (body weight ≤100 kg and total insulin dose <1.2 units/kg/day). Participants received a single dose of either AT278 U500 or IAsp U100 (0.3 units/kg s.c.) in a crossover design, followed by an 8-h euglycemic clamp in the absence of basal insulin. RESULTS: With AT278 U500, onset of appearance in serum was 6 min earlier (P < 0.0001) and reached 50% of maximum concentration 23 min faster (P < 0.0001). Insulin exposure with AT278 U500 was 4.0-fold higher within the first 30 min (95% CI 3.29, 4.90), 1.5-fold higher within the first 60 min (95% CI 1.35, 1.76), and statistically superior up to 90 min postdose (P < 0.05). With AT278 U500, onset of action was 10 min earlier (P < 0.0001) and reached 50% of maximum glucose infusion rate 20 min faster (P < 0.0001). The glucose-lowering effect with AT278 U500 was 8.9-fold higher within the first 30 min (95% CI 5.96, 17.46), 2.4-fold higher within the first 60 min (95% CI 1.92, 3.22), and statistically superior up to 2 h postdose (P < 0.0001). Overall insulin exposure and glucose-lowering effect were comparable. No significant safety findings were observed. CONCLUSIONS: AT278 U500 offers rapid-acting characteristics in a reduced dose volume, with accelerated absorption and onset of action compared with IAsp U100 in the studied population.

REC drives recombination to repair double-strand breaks in animal mtDNA.

Mechanisms that safeguard mitochondrial DNA (mtDNA) limit the accumulation of mutations linked to mitochondrial and age-related diseases. Yet, pathways that repair double-strand breaks (DSBs) in animal mitochondria are poorly understood. By performing a candidate screen for mtDNA repair proteins, we identify that REC-an MCM helicase that drives meiotic recombination in the nucleus-also localizes to mitochondria in Drosophila. We show that REC repairs mtDNA DSBs by homologous recombination in somatic and germline tissues. Moreover, REC prevents age-associated mtDNA mutations. We further show that MCM8, the human ortholog of REC, also localizes to mitochondria and limits the accumulation of mtDNA mutations. This study provides mechanistic insight into animal mtDNA recombination and demonstrates its importance in safeguarding mtDNA during ageing and evolution.

Rapid identification of pathogens in blood serum via Raman tweezers in combination with advanced processing methods.

Pathogenic microbes contribute to several major global diseases that kill millions of people every year. Bloodstream infections caused by these microbes are associated with high morbidity and mortality rates, which are among the most common causes of hospitalizations. The search for the "Holy Grail" in clinical diagnostic microbiology, a reliable, accurate, low cost, real-time, and easy-to-use diagnostic method, is one of the essential issues in clinical practice. These very critical conditions can be met by Raman tweezers in combination with advanced analysis methods. Here, we present a proof-of-concept study based on Raman tweezers combined with spectral mixture analysis that allows for the identification of microbial strains directly from human blood serum without user intervention, thus eliminating the influence of a data analyst.

In Vivo Contrast Imaging of Rat Heart with Carbon Dioxide Foam.

Widely used classical angiography with the use of iodine contrast agents is highly problematic, particularly in patients with diabetes mellitus, cardiac and pulmonary diseases, or degree III or IV renal insufficiency. Some patients may be susceptible to allergic reaction to the iodine contrast substance. The intravenous injection of a bolus of CO2 (negative contrast) is an alternative method, which is, however, currently only used for imaging blood vessels of the lower limbs. The aim of our project was to design and test on an animal model a methodology for injecting the CO2 foam which would minimize the possibility of embolization of the brain tissue and heart infarction, leading to their damage. This is important research for the further promotion of the use of CO2, which is increasingly important for endovascular diagnosis and treatment, because carbon-dioxide-related complications are extremely rare. CO2 foam was prepared by the rapid mixing in a 2:1 ratio of CO2 and fetal bovine serum (FBS)-enriched Dulbecco's Modified Eagle Medium (DMEM). Freshly prepared CO2 foam was administered into the catheterized rat tail vein or cannulated rat abdominal aorta and inferior vena cava (IVC). CO2 foam was compared with commercially available microbubbles (lipid shell/gas core). The rat heart in its parasternal long axis was imaged in B-Mode and Non-linear Contrast Mode before/during and after the contrast administration. Samples of the brain, heart and lungs were collected and subjected to histological examination. The non-linear contrast imaging method enables the imaging of micron-sized gas microbubbles inside a rat heart. The significantly shorter lifetime of the prepared CO2 foam is a benefit for avoiding the local ischemia of tissues.

Aberrant cyclin C nuclear release induces mitochondrial fragmentation and dysfunction in MED13L syndrome fibroblasts.

MED13L syndrome is a haploinsufficiency developmental disorder characterized by intellectual disability, heart malformation, and hypotonia. MED13L controls transcription by tethering the cyclin C-Cdk8 kinase module (CKM) to the Mediator complex. In addition, cyclin C has CKM-independent roles in the cytoplasm directing stress-induced mitochondrial fragmentation and regulated cell death. Unstressed MED13L S1497 F/fs patient fibroblasts exhibited aberrant cytoplasmic cyclin C localization, mitochondrial fragmentation, and a 6-fold reduction in respiration. In addition, the fibroblasts exhibited reduced mtDNA copy number, reduction in mitochondrial membrane integrity, and hypersensitivity to oxidative stress. Finally, transcriptional analysis of MED13L mutant fibroblasts revealed reduced mRNA levels for several genes necessary for normal mitochondrial function. Pharmacological or genetic approaches preventing cyclin C-mitochondrial localization corrected the fragmented mitochondrial phenotype and partially restored organelle function. In conclusion, this study found that mitochondrial dysfunction is an underlying defect in cells harboring the MED13L S1497 F/fs allele and identified cyclin C mis-localization as the likely cause. These results provide a new avenue for understanding this disorder.

Raman spectroscopy-a tool for rapid differentiation among microbes causing urinary tract infections.

Urinary tract infections belong to the most common infections in the world. Besides community-acquired infections, nosocomial infections pose a high risk especially for patients having indwelling catheters, undergoing urological surgeries or staying at hospital for prolonged time. They can be often complicated by antimicrobial resistance and/or biofilm formation. Therefore, a rapid diagnostic tool enabling timely identification of a causative agent and its susceptibility to antimicrobials is a need. Raman spectroscopy appears to be a suitable method that allows rapid differentiation among microbes and provides a space for further analyses, such as determination of capability of biofilm formation or antimicrobial susceptibility/resistance in tested strains. Our work here presents a possibility to differ among most common microbes causing urinary tract infections (belonging to 20 species). We tested 254 strains directly from colonies grown on Mueller-Hinton agar plates. The results show that it is possible to distinguish among the tested species using Raman spectroscopy, which proves its great potential for future use in clinical diagnostics. Moreover, we present here a pilot study of a real-time analysis and identification (in less than 10 min) of single microbial cells directly in urine employing optical tweezers combined with Raman spectroscopy.

Optically Transportable Optofluidic Microlasers with Liquid Crystal Cavities Tuned by the Electric Field.

Liquid crystal microdroplets with readily adjustable optical properties have attracted considerable attention for building reconfigurable optofluidic microsystems for sensing, imaging, and light routing applications. In this quest, development of active optical microcavities serving as versatile integrated sources of coherent light and ultra-sensitive environmental sensors has played a prominent role. Here, we study transportable optofluidic microlasers reversibly tunable by an external electric field, which are based on fluorophore-doped emulsion droplets of radial nematic liquid crystals manipulated by optical tweezers in microfluidic chips with embedded liquid electrodes. Full transparency of the electrodes formed by a concentrated electrolyte solution allows for applying an electric field to the optically trapped droplets without undesired heating caused by light absorption. Taking advantage of independent, precise control over the electric and thermal stimulation of the lasing liquid crystal droplets, we characterize their spectral tuning response at various optical trapping powers and study their relaxation upon a sudden decrease in the trapping power. Finally, we demonstrate that sufficiently strong applied electric fields can induce fully reversible phase transitions in the trapped droplets even below the bulk melting temperature of the used liquid crystal. Our observations indicate viability of creating electrically tunable, optically transported microlasers that can be prepared on-demand and operated within microfluidic chips to implement integrated microphotonic or sensing systems.

Psychodidae (Diptera) of Azerbaijan and Georgia - faunistics with biodiversity notes.

Records of 46 Psychodidae (Sycoracinae 1, Trichomyiinae 1, Psychodinae 44) species/subspecies are presented in this paper based on specimens collected by sweep-netting in Azerbaijan and Georgia in 2019. Nine species are recorded for the first time since their original description; 12 species are new for Transcaucasia; 22 species are new for Azerbaijan; and 17 species are new for Georgia. Saraiella ressli montana Jezek, 1990 is proposed as a synonym of S. ressli Wagner, 1983, syn. nov. Knowledge of some aspects of the ecology and biogeography of selected (especially rare) species has been expanded and a clear pattern was found in species richness, rare species, and new records in relation to land use, habitat diversity, and preservation of the environment surrounding the sampling site.

Two new Palaearctic species of moth flies (Diptera, Psychodidae, Psychodinae) from the Caucasus Mts.

Psychodid specimens were sorted from samples collected during fieldwork in areas in and around the Caucasus Mountains in 2019. Thornburghiella montana sp. nov. (from Georgia) and Pericoma inopinata sp. nov. (from Azerbaijan and Georgia) are described. Differential diagnoses are given and diagnostic characters illustrated.

Raman Microspectroscopic Analysis of Selenium Bioaccumulation by Green Alga Chlorella vulgaris.

Selenium (Se) is an element with many commercial applications as well as an essential micronutrient. Dietary Se has antioxidant properties and it is known to play a role in cancer prevention. However, the general population often suffers from Se deficiency. Green algae, such as Chlorella vulgaris, cultivated in Se-enriched environment may be used as a food supplement to provide adequate levels of Se. We used Raman microspectroscopy (RS) for fast, reliable, and non-destructive measurement of Se concentration in living algal cells. We employed inductively coupled plasma-mass spectrometry as a reference method to RS and we found a substantial correlation between the Raman signal intensity at 252 cm-1 and total Se concentration in the studied cells. We used RS to assess the uptake of Se by living and inactivated algae and demonstrated the necessity of active cellular transport for Se accumulation. Additionally, we observed the intracellular Se being transformed into an insoluble elemental form, which we further supported by the energy-dispersive X-ray spectroscopy imaging.

The Impact of Mitochondrial Fission-Stimulated ROS Production on Pro-Apoptotic Chemotherapy.

Cancer is one of the world's deadliest afflictions. Despite recent advances in diagnostic and surgical technologies, as well as improved treatments of some individual tumor types, there is currently no universal cure to prevent or impede the uncontrolled proliferation of malignant cells. Targeting tumors by inducing apoptosis is one of the pillars of cancer treatment. Changes in mitochondrial morphology precede intrinsic apoptosis, but mitochondrial dynamics has only recently been recognized as a viable pharmacological target. In many cancers, oncogenic transformation is accompanied by accumulation of elevated cellular levels of ROS leading to redox imbalance. Hence, a common chemotherapeutic strategy against such tumor types involves deploying pro-oxidant agents to increase ROS levels above an apoptotic death-inducing threshold. The aim of this chapter is to investigate the benefit of stimulating mitochondrial fission-dependent production of ROS for enhanced killing of solid tumors. The main question to be addressed is whether a sudden and abrupt change in mitochondrial shape toward the fragmented phenotype can be pharmacologically harnessed to trigger a burst of mitochondrial ROS sufficient to initiate apoptosis specifically in cancer cells but not in non-transformed healthy tissues.

Pharmacokinetics and Pharmacodynamics of Three Different Formulations of Insulin Aspart: A Randomized, Double-Blind, Crossover Study in Men With Type 1 Diabetes.

OBJECTIVE: To investigate the pharmacokinetic and pharmacodynamic properties and safety of a novel formulation of insulin aspart (AT247) versus two currently marketed insulin aspart formulations (NovoRapid [IAsp] and Fiasp [faster IAsp]). RESEARCH DESIGN AND METHODS: This single-center, randomized, double-blind, three-period, crossover study was conducted in 19 men with type 1 diabetes, receiving single dosing of trial products (0.3 units/kg) in a random order on three visits. Pharmacokinetics and pharmacodynamics were assessed during a euglycemic clamp lasting up to 8 h. RESULTS: Onset of insulin appearance was earlier for AT247 compared with IAsp (-12 min [95% CI -14; -8], P = 0.0004) and faster IAsp (-2 min [-5; -2], P = 0.0003). Onset of action was accelerated compared with IAsp (-23 min [-37; -15], P = 0.0004) and faster IAsp (-9 min [-11; -3], P = 0.0006). Within the first 60 min, a higher exposure was observed for AT247 compared with IAsp by the area under the curve (AUC) glucose infusion rate (GIR) from 0 to 60 min (AUCAsp0-60min: treatment ratio vs. IAsp 2.3 [1.9; 2.9] vs. faster IAsp 1.5 [1.3; 1.8]), which was underpinned by a greater early glucose-lowering effect (AUCGIR,0-60min: treatment ratio vs. IAsp 2.8 [2.0; 5.5] vs. faster IAsp 1.7 [1.3; 2.3]). Furthermore, an earlier offset of exposure was observed for AT247 compared with IAsp (-32 min [-58; -15], P = 0.0015) and faster IAsp (-27 min [-85; -15], P = 0.0017), while duration of the glucose-lowering effect, measured by time to late half-maximum effect, did not differ significantly. CONCLUSIONS: AT247 exhibited an earlier insulin appearance, exposure, and offset, with corresponding enhanced early glucose-lowering effect compared with IAsp and faster IAsp. It therefore represents a promising candidate in the pursuit for second-generation prandial insulin analogs to improve postprandial glycemic control.

Redescription of Armillipora Quate (Diptera: Psychodidae: Psychodinae) with a new species from Bolivia.

The former monotypic genus Armillipora Quate, known only from Costa Rica and Panama, is redescribed, including the type species A. selvica Quate, this time collected on the Caribbean side of Nicaragua, RAAN department, and illustrated based on male morphological characters. The male of a new species, A. suapiensis sp. nov., from Bolivia, La Paz department, is described here and also figured.

Synopsis of the Psychodidae (Diptera) fauna of Bulgaria.

Records of 81 Psychodidae (Sycoracinae 4 spp., Psychodinae 76 spp.) species/subspecies are presented in this paper based on specimens collected in Bulgaria. 46 species are new records for Bulgaria (Sycoracinae 3 spp., Psychodinae 43 spp.). The Psychodidae fauna of Bulgaria now comprises 99 species (Phlebotominae 5 spp., Sycoracinae 5 spp., Trichomyiinae 1 sp., and Psychodinae 88 spp.).