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The extracellular glycerol kinase gene from Saccharomyces cerevisiae (GUT1) was cloned into the expression vector pPICZα A and integrated into the genome of the methylotrophic yeast Pichia pastoris X-33. The presence of the GUT1 insert was confirmed by PCR analysis. Four clones were selected and the functionality of the recombinant enzyme was assayed. Among the tested clones, one exhibited glycerol kinase activity of 0.32 U/mL, with specific activity of 0.025 U/mg of protein. A medium optimized for maximum biomass production by recombinant Pichia pastoris in shaker cultures was initially explored, using 2.31 % (by volume) glycerol as the carbon source. Optimization was carried out by response surface methodology (RSM). In preliminary experiments, following a Plackett-Burman design, glycerol volume fraction (φ(Gly)) and growth time (t) were selected as the most important factors in biomass production. Therefore, subsequent experiments, carried out to optimize biomass production, followed a central composite rotatable design as a function of φ(Gly) and time. Glycerol volume fraction proved to have a significant positive linear effect on biomass production. Also, time was a significant factor (at linear positive and quadratic levels) in biomass production. Experimental data were well fitted by a convex surface representing a second order polynomial model, in which biomass is a function of both factors (R²=0.946). Yield and specific activity of glycerol kinase were mainly affected by the additions of glycerol and methanol to the medium. The optimized medium composition for enzyme production was: 1 % yeast extract, 1 % peptone, 100 mM potassium phosphate buffer, pH=6.0, 1.34 % yeast nitrogen base (YNB), 4·10^–5 % biotin, 1 % methanol and 1 % glycerol, reaching 0.89 U/mL of glycerol kinase activity and 14.55 g/L of total protein in the medium after 48 h of growth.

Abstract Background In longitudinal health services research, hospital identification using an ID code, often supplemented with several additional variables, lacks clarity regarding representativeness and variable influence. This study presents an operational method for hospital identity delimitation and a novel longitudinal identification approach, demonstrated using a case study. Methods The conceptualisation considers hospitals as evolving entities, identifying “similar enough” pairs across two time points using an automated similarity matrix. This method comprises key variable selection, similarity scoring, and tolerance threshold definition, tailored to data source characteristics and clinical relevance. This linking method is tested by applying the identification of minimum caseload requirements-related German hospitals, utilizing German Hospital Quality Reports (GHQR) 2016–2020. Results The method achieved a success rate (min: 97.9% - max: 100%, mean: 99.9%) surpassing traditional hospital ID-code linkage (min: 91.5% - max: 98.8%, mean: 96.6%), with a remarkable 99% reduction in manual work through automation. Conclusions This method, rooted in a comprehensive understanding of hospital identities, offers an operational, automated, and customisable process serving diverse clinical topics. This approach has the advantage of simultaneously considering multiple variables and systematically observing temporal changes in hospitals. It also enhances the precision and efficiency of longitudinal hospital identification in health services research.

Abstract Expansion of the glutamine tract (poly-Q) in the protein huntingtin (HTT) causes the neurodegenerative disorder Huntington’s disease (HD). Emerging evidence suggests that mutant HTT (mHTT) disrupts brain development. To gain mechanistic insights into the neurodevelopmental impact of human mHTT, we engineered male induced pluripotent stem cells to introduce a biallelic or monoallelic mutant 70Q expansion or to remove the poly-Q tract of HTT. The introduction of a 70Q mutation caused aberrant development of cerebral organoids with loss of neural progenitor organization. The early neurodevelopmental signature of mHTT highlighted the dysregulation of the protein coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2), a transcription factor involved in mitochondrial integrated stress response. CHCHD2 repression was associated with abnormal mitochondrial morpho-dynamics that was reverted upon overexpression of CHCHD2. Removing the poly-Q tract from HTT normalized CHCHD2 levels and corrected key mitochondrial defects. Hence, mHTT-mediated disruption of human neurodevelopment is paralleled by aberrant neurometabolic programming mediated by dysregulation of CHCHD2, which could then serve as an early interventional target for HD.

La tecnología Long Range (LoRa), mediante el protocolo LoRaWAN, permite la transmisión eficiente de datos a largas distancias con mínimos recursos, funcionando así por un tiempo más prolongado. Esta tecnología es utilizada mayormente para la monitorización y puede ser utilizada en una gran cantidad de áreas que van desde la meteorología hasta aplicaciones médicas. Se realizó un estudio de cobertura en algunos sectores de la Universidad de El Salvador entre los meses de noviembre y diciembre, donde se buscó establecer una metodología para predecir el nivel de recepción en diversas condiciones geográficas dentro de esta. Para ello, se utilizaron diversos recursos como softwares de simulación, modelos matemáticos, mediciones de campo con el analizador de espectro RSA306B de la marca Tektronix y la placa TTGO LoRa 32, para determinar su eficacia bajo condiciones específicas. Los datos se tabulan, comparan y se elige la herramienta más adecuada de acuerdo con el entorno, Los cuales van desde zonas con gran presencia de edificaciones a zonas con densa vegetación.

Across the animal kingdom, neural responses in the auditory cortex are suppressed during vocalization, and humans are no exception. A common hypothesis is that suppression increases sensitivity to auditory feedback, enabling the detection of vocalization errors. This hypothesis has been previously confirmed in non-human primates, however a direct link between auditory suppression and sensitivity in human speech monitoring remains elusive. To address this issue, we obtained intracranial electroencephalography (iEEG) recordings from 35 neurosurgical participants during speech production. We first characterized the detailed topography of auditory suppression, which varied across superior temporal gyrus (STG). Next, we performed a delayed auditory feedback (DAF) task to determine whether the suppressed sites were also sensitive to auditory feedback alterations. Indeed, overlapping sites showed enhanced responses to feedback, indicating sensitivity. Importantly, there was a strong correlation between the degree of auditory suppression and feedback sensitivity, suggesting suppression might be a key mechanism that underlies speech monitoring. Further, we found that when participants produced speech with simultaneous auditory feedback, posterior STG was selectively activated if participants were engaged in a DAF paradigm, suggesting that increased attentional load can modulate auditory feedback sensitivity.

ABSTRACT While bioactivity and a favorable safety profile for biotherapeutics is of utmost importance, manufacturability is also worth of consideration to ease the manufacturing process. Manufacturability in the scientific literature is mostly related to stability of formulated drug substances, with limited focus on downstream process‐related manufacturability, that is, how easily can a protein be purified. Process‐related impurities or biological impurities like viruses and host cell proteins (HCP) are present in the harvest which have mostly acid isoelectric points and need to be removed to ensure patient safety. Therefore, during molecule design, the surface charge of the target molecule should preferably differ sufficiently from the surface charge of the impurities to enable an efficient purification strategy. In this feasibility study, we evaluated the possibility of improving manufacturability by adapting the surface charge of the target protein. We generated several variants of a GLP1‐receptor‐agonist‐Fc‐domain‐FGF21‐fusion protein and demonstrated proof of concept exemplarily for an anion exchange chromatography step which then can be operated at high pH values with maximal product recovery allowing removal of HCP and viruses. Altering the surface charge distribution of biotherapeutic proteins can thus be useful allowing for an efficient manufacturing process for removing HCP and viruses, thereby reducing manufacturing costs.