Statistical Experimental Designs for cLTB-Syn Vaccine Production Using Daucus carota Cell Suspension Cultures.

The carrot-made LTB-Syn antigen (cLTB-Syn) is a vaccine candidate against synucleinopathies based on carrot cells expressing the target antigen LTB and syn epitopes. Therefore, the development of an efficient production process is required with media culture optimization to increase the production yields as the main goal. In this study, the effect of two nitrogen sources (urea and glutamate) on callus cultures producing cLTB-Syn was studied, observing that the addition of 17 mM urea to MS medium favored the biomass yield. To optimize the MS media composition, the influence of seven medium components on biomass and cLTB-Syn production was first evaluated by a Plackett-Burman design (PBD). Then, three factors were further analyzed using a central composite design (CCD) and response surface methodology (RSM). The results showed a 1.2-fold improvement in biomass, and a 4.5-fold improvement in cLTB-Syn production was achieved at the shake-flask scale. At the bioreactor scale, there was a 1.5-fold increase in biomass and a 2.8-fold increase in cLTB-Syn yield compared with the standard MS medium. Moreover, the cLTB-Syn vaccine induced humoral responses in BALB/c mice subjected to either oral or subcutaneous immunization. Therefore, cLTB-Syn is a promising vaccine candidate that will aid in developing immunotherapeutic strategies to combat PD and other neurodegenerative diseases without the need for cold storage, making it a financially viable option for massive immunization.

Establishment of the Daucus carota SMC-1 Cell Suspension Line for Poliovirus Vaccine Development.

The development of virus-free, oral vaccines against poliovirus capable of inducing mucosal protective immunity is needed to safely combat this pathogen. In the present study, a carrot cell line expressing the poliovirus VP2 antigen was established at the level of callus and cell suspensions, exploring the effects of culture media (MS and B5), supplementation with urea, phytoregulators (2,4-D : KIN), and light conditions (continuous light, photoperiod, and total darkness). The best callus growth was obtained on B5 medium supplemented with 2 mg/L of 2,4-D + 2 mg/L kinetin and 0.0136 g/L of urea and in continuous light conditions. Suspension cultures of the SMC-1 line in 250 mL Erlenmeyer flasks had a maximum growth of 16.07 ± 0.03 g/L DW on day 12 with a growth rate of µ=0.3/d and a doubling time of 2.3 days. In a 2 L airlift bioreactor, the biomass yield achieved was 25.6 ± 0.05 g/L DW at day 10 with a growth rate of µ= 0.58/d and doubling time of 1.38 d. Cell growth was 1.5 times higher in bioreactors than in shake flasks, highlighting that both systems resulted in the accumulation of VP2 throughout the time in culture. The maximum VP2 yield in flasks was 387.8 µg/g DW at day 21, while in the reactor it was 550.2 µg/g DW at day 18. In conclusion, bioreactor-based production of the VP2 protein by the SMC-1 suspension cell line offers a higher productivity when compared to flask cultures, offering a key perspective to produce low-cost vaccines against poliomyelitis.

Stress granules plug and stabilize damaged endolysosomal membranes.

Endomembrane damage represents a form of stress that is detrimental for eukaryotic cells1,2. To cope with this threat, cells possess mechanisms that repair the damage and restore cellular homeostasis3-7. Endomembrane damage also results in organelle instability and the mechanisms by which cells stabilize damaged endomembranes to enable membrane repair remains unknown. Here, by combining in vitro and in cellulo studies with computational modelling we uncover a biological function for stress granules whereby these biomolecular condensates form rapidly at endomembrane damage sites and act as a plug that stabilizes the ruptured membrane. Functionally, we demonstrate that stress granule formation and membrane stabilization enable efficient repair of damaged endolysosomes, through both ESCRT (endosomal sorting complex required for transport)-dependent and independent mechanisms. We also show that blocking stress granule formation in human macrophages creates a permissive environment for Mycobacterium tuberculosis, a human pathogen that exploits endomembrane damage to survive within the host.

Production and characterization of a chimeric protein targeting synuclein epitopes for immunotherapy against synucleinopathies.

The aggregation and spread of alpha-synuclein (αSyn) is associated with several pathogenic pathways that lead to neurodegeneration and, ultimately, to synucleinopathies development. Hence, the establishment of a safe and effective disease-modifying therapy that limits or prevents the spread of toxic αSyn aggregation could lead to positive clinical outcomes. A rational vaccine design can be focused on the selection of specific epitopes able to induce the immune response desired, for example, antibodies able to mediate the clearance of αSyn aggregates without the induction of inflammatory responses. To develop a rapid system for the evaluation of a vaccine candidate against synucleinopathies, rLTB-Syn (an antigen based on three B cell epitopes from αSyn and the B subunit of the heat-labile Escherichia coli enterotoxin [LTB] as adjuvant/carrier) was produced using recombinant E. coli (Rosetta DE3) as the expression host. The bacterial version of rLTB-Syn was produced as soluble protein at yields up to 1.72 mg/g biomass. A method for the purification of rLTB-Syn (~18 kDa) was developed based on ion exchange chromatography, reaching purity >93% with a final concentration of 82.6 µg/mL. Furthermore, the purified soluble rLTB-Syn retained GM1 binding activity, suggesting proper folding and pentameric structure. The results from this study establish a fast and effective method to obtain rLTB-Syn, making it useful in the design of novel vaccine formulations targeting synucleinopathies.

Prevalence of SARS-CoV-2 Variants and Disease Outcome of COVID-19 Patients in the Amazonas Region of Peru.

Peru was severely affected by COVID-19 with a fatality rate that reached up to 6%. In this study, the relationship between SARS-CoV-2 variants and COVID-19 disease outcome in Amazonas, a region of northeastern Peru, was evaluated. The variants were determined by genomic sequencing, and clinical-epidemiological data were collected from 590 patients between April 2021 and February 2022. There was no association between mortality and hospitalization with any of the variants, but we did find that Omicron is more likely to infect vaccinated and nonvaccinated people. A significant association was also found between unvaccinated patients and hospitalization. Interestingly, in the indigenous population, there were fewer hospitalizations than in the general population. In conclusion, SARS-CoV-2 variants were not associated with the disease outcome in the Amazonas region, and indigenous population were found to be less vulnerable to severe COVID-19 illness.

Primer reporte de un brote de dengue en balsas, Amazonas, Perú, durante 2021 y 2022

Introducción: El aumento de casos de dengue en Amazonas es un riesgo para la salud pública. En el 2021, Balsas reportó por primera vez un brote de dengue. Métodos: La población incluyó a pacientes que cumplían con la definición de caso entre diciembre 2021 y febrero 2022. La identificación de los serotipos se determinó mediante una qRT-PCR múltiplex. Resultados: Se identificaron 72 pacientes de los cuales 53 (74%) se confirmaron por serología (Ag NS1). El serotipo prevalente fue DENV-2 (94%), y el 6% fue DENV-1. Los pacientes de 19 a 45 años presentaron el mayor porcentaje de casos (59%). Los síntomas más frecuentes fueron cefalea, mialgias, fiebre y artralgias; el 23 % presentó dolor abdominal intenso. Conclusión: Este fue el primer brote de dengue confirmado en el distrito de Balsas, siendo DENV-2 el principal causante, destacando la necesidad de mejorar la vigilancia en zonas sin transmisión autóctona de la enfermedad.

Introduction: The increase in dengue cases in Amazonas represents a public health risk. In 2021, Balsas reported a dengue outbreak for the first time. Methods: The population included patients who met the case definition between December 2021 and February 2022. Serotype identification was determined using a multiplex qRT-PCR. Results: A total of 72 patients were identified, of which 53 (74%) were confirmed by serology (NS1 Ag). The prevalent serotype was DENV-2 (94%), and 6% were DENV-1. Patients aged 19 to 45 years had the highest percentage of cases (59%). The most frequent symptoms were headache, myalgia, fever, and arthralgia; 23% had intense abdominal pain. Conclusion: This was the first confirmed dengue outbreak in the Balsas district, with DENV-2 being the main cause of the outbreak, highlighting the need to improve surveillance in areas without autochthonous transmission of the disease.

Modelling membrane reshaping by staged polymerization of ESCRT-III filaments.

ESCRT-III filaments are composite cytoskeletal polymers that can constrict and cut cell membranes from the inside of the membrane neck. Membrane-bound ESCRT-III filaments undergo a series of dramatic composition and geometry changes in the presence of an ATP-consuming Vps4 enzyme, which causes stepwise changes in the membrane morphology. We set out to understand the physical mechanisms involved in translating the changes in ESCRT-III polymer composition into membrane deformation. We have built a coarse-grained model in which ESCRT-III polymers of different geometries and mechanical properties are allowed to copolymerise and bind to a deformable membrane. By modelling ATP-driven stepwise depolymerisation of specific polymers, we identify mechanical regimes in which changes in filament composition trigger the associated membrane transition from a flat to a buckled state, and then to a tubule state that eventually undergoes scission to release a small cargo-loaded vesicle. We then characterise how the location and kinetics of polymer loss affects the extent of membrane deformation and the efficiency of membrane neck scission. Our results identify the near-minimal mechanical conditions for the operation of shape-shifting composite polymers that sever membrane necks.

Malaria y COVID-19 en comunidades nativas de Amazonas, Perú / Malaria and COVID-19 in native communities of Amazonas, Peru

Introducción En los últimos años, el número de casos de malaria en las comunidades nativas de Condorcanqui, Amazonas ha aumentado considerablemente. La malaria por Plasmodium vivax es endémica en la región y en 2019 fue reportada la reintroducción de P. falciparum. Métodos En este estudio, recopilamos y analizamos los datos de malaria y COVID-19 reportados por la Dirección Regional de Salud (DIRESA) durante el 2020. Además, realizamos un análisis de razón de posibilidades "odds ratio" para evaluar las asociaciones significativas entre los síntomas de la COVID-19 y las infecciones previas de malaria. Resultados En el 2020, se reportaron 1547 casos de malaria (97% por P. vivax) y 5968 de COVID-19. Por otro lado, 96 pacientes contrajeron COVID-19 después de contraer una infección de malaria. De éstos, 87 eran sintomáticos (90,6%) y en su mayoría adultos de 30 a 59 años (62,3%). Además, encontramos que las infecciones previas de malaria están asociadas a la presencia de síntomas como fiebre, tos, dolor de garganta y dificultad respiratoria. Sin embargo, no hubo asociación significativa entre estos casos y la hospitalización o la muerte. Conclusión Nuestro análisis sugiere que las infecciones previas por malaria podrían afectar la sintomatología de la COVID-19, lo que destaca la importancia de un programa continuo de control y vigilancia de la malaria para evitar posibles sindemias con la COVID-19.

Introduction In recent years, the number of malaria cases in native communities from Condorcanqui, Amazonas has considerably increased. Plasmodium vivax malaria is endemic in the region and the re-introduction of P. falciparum was reported in 2019. Methods Here, we compiled and analyzed malaria and COVID-19 data reported by the Regional Direction of Health (DIRESA) during the 2020. Additionally, we performed an odds ratio analysis to evaluate significant associations between COVID-19 symptoms and previous malaria infections. Results In 2020, 1547 malaria (97% were P. vivax) and 5968 COVID-19 cases were reported. Furthermore, 96 patients got COVID-19 after getting a malaria infection. From these, 87 were symptomatic (90.6%), and mostly adults, ages 30 to 59 (62.3%). Also, we found that malaria previous infections represent a risk for the presence of symptoms such as fever, cough, throat pain, and respiratory difficulty. Nevertheless, there was no significant association between these cases and hospitalization or death. Conclusion Our analysis suggests that previous malaria infections might affect COVID-19 symptomatology, which highlights the importance of a continuing control and surveillance malaria program to avoid potential syndemics with COVID-19.

Physical mechanisms of ESCRT-III-driven cell division.

Living systems propagate by undergoing rounds of cell growth and division. Cell division is at heart a physical process that requires mechanical forces, usually exerted by assemblies of cytoskeletal polymers. Here we developed a physical model for the ESCRT-III-mediated division of archaeal cells, which despite their structural simplicity share machinery and evolutionary origins with eukaryotes. By comparing the dynamics of simulations with data collected from live cell imaging experiments, we propose that this branch of life uses a previously unidentified division mechanism. Active changes in the curvature of elastic cytoskeletal filaments can lead to filament perversions and supercoiling, to drive ring constriction and deform the overlying membrane. Abscission is then completed following filament disassembly. The model was also used to explore how different adenosine triphosphate (ATP)-driven processes that govern the way the structure of the filament is changed likely impact the robustness and symmetry of the resulting division. Comparisons between midcell constriction dynamics in simulations and experiments reveal a good agreement with the process when changes in curvature are implemented at random positions along the filament, supporting this as a possible mechanism of ESCRT-III-dependent division in this system. Beyond archaea, this study pinpoints a general mechanism of cytokinesis based on dynamic coupling between a coiling filament and the membrane.

Establishment of the Carrot-Made LTB-Syn Antigen Cell Line in Shake Flask and Airlift Bioreactor Cultures.

Carrot (Daucus carota) cells have been used to effectively manufacture recombinant biopharmaceuticals such as cytokines, vaccines, and antibodies. We generated the carrot cell line Z4, genetically modified to produce the LTB-Syn antigen, which is a fusion protein proposed for immunotherapy against synucleinopathies. In this work, the Z4 cell suspension line was cultivated to produce the LTB-Syn protein in a 250 mL shake flask and 2 L airlift bioreactor cultures grown for 45 and 30 days, respectively. Maximum biomass was obtained on day 15 in both the airlift bioreactor (35.00 ± 0.04 g/L DW) and shake flasks (17.00 ± 0.04 g/L DW). In the bioreactor, the highest LTB-Syn protein yield (1.52 ± 0.03 µg/g FW) was obtained on day 15; while the same occurred on day 18 for shake flasks (0.92 ± 0.02 µg/g FW). LTB-Syn protein levels were analyzed by GM1-ELISA and western blot. PCR analysis confirmed the presence of the transgene in the Z4 line. The obtained data demonstrate that the carrot Z4 cell suspension line grown in airlift bioreactors shows promise for a scale-up cultivation producing an oral LTB-Syn antigen.

Collective motion of run-and-tumble repulsive and attractive particles in one-dimensional systems.

Active matter deals with systems whose particles consume energy at the individual level in order to move. To unravel features such as the emergence of collective structures, several models have been suggested, such as the on-lattice model of run-and-tumble particles implemented via the persistent exclusion process (PEP). In our work, we study a one-dimensional system of run-and-tumble repulsive or attractive particles, both on-lattice and off-lattice. Additionally, we implement cluster motility dynamics in the on-lattice case (since in the off-lattice case, cluster motility arises from the individual particle dynamics). While we observe important differences between discrete and continuous dynamics, few common features are of particular importance. Increasing particle density drives aggregation across all different systems explored. For non-attractive particles, the effects of particle activity on aggregation are largely independent of the details of the dynamics. In contrast, once attractive interactions are introduced, the steady-state, which is completely determined by the interplay between these and the particles' activity, becomes highly dependent on the details of the dynamics.

Modelling the dynamics of vesicle reshaping and scission under osmotic shocks.

We study the effects of osmotic shocks on lipid vesicles via coarse-grained molecular dynamics simulations by explicitly considering the solute in the system. We find that depending on their nature (hypo- or hypertonic) such shocks can lead to bursting events or engulfing of external material into inner compartments, among other morphology transformations. We characterize the dynamics of these processes and observe a separation of time scales between the osmotic shock absorption and the shape relaxation. Our work consequently provides an insight into the dynamics of compartmentalization in vesicular systems as a result of osmotic shocks, which can be of interest in the context of early proto-cell development and proto-cell compartmentalisation.

Influence of membrane-cortex linkers on the extrusion of membrane tubes.

The cell membrane is an inhomogeneous system composed of phospholipids, sterols, carbohydrates, and proteins that can be directly attached to underlying cytoskeleton. The protein linkers between the membrane and the cytoskeleton are believed to have a profound effect on the mechanical properties of the cell membrane and its ability to reshape. Here, we investigate the role of membrane-cortex linkers on the extrusion of membrane tubes using computer simulations and experiments. In simulations, we find that the force for tube extrusion has a nonlinear dependence on the density of membrane-cortex attachments: at a range of low and intermediate linker densities, the force is not significantly influenced by the presence of the membrane-cortex attachments and resembles that of the bare membrane. For large concentrations of linkers, however, the force substantially increases compared with the bare membrane. In both cases, the linkers provided membrane tubes with increased stability against coalescence. We then pulled tubes from HEK cells using optical tweezers for varying expression levels of the membrane-cortex attachment protein Ezrin. In line with simulations, we observed that overexpression of Ezrin led to an increased extrusion force, while Ezrin depletion had a negligible effect on the force. Our results shed light on the importance of local protein rearrangements for membrane reshaping at nanoscopic scales.

Surveillance of antimicrobial resistance in Serratia marcescens in Mexico.

Antimicrobial resistance is a global public health threat. Therefore, surveillance studies are important tools to help direct antimicrobial use. The aim of this study was to investigate antimicrobial resistance in Serratia marcescens isolates collected in 2016-2017 at eight medical centers from two regions of Mexico. Selected S. marcescens isolates were further tested by polymerase chain reaction to detect the presence of genes encoding the ß-lactamases, SHV, TEM or CTX. Antimicrobial resistance continues to be high in Mexico, particularly to ciprofloxacin and aminoglycosides. Also, a widespread prevalence of blaTEM was detected in S. marcescens isolates.

Brain Ventricular System and Cerebrospinal Fluid Development and Function: Light at the End of the Tube: A Primer with Latest Insights.

The brain ventricular system is a series of connected cavities, filled with cerebrospinal fluid (CSF), that forms within the vertebrate central nervous system (CNS). The hollow neural tube is a hallmark of the chordate CNS, and a closed neural tube is essential for normal development. Development and function of the ventricular system is examined, emphasizing three interdigitating components that form a functional system: ventricle walls, CSF fluid properties, and activity of CSF constituent factors. The cellular lining of the ventricle both can produce and is responsive to CSF. Fluid properties and conserved CSF components contribute to normal CNS development. Anomalies of the CSF/ventricular system serve as diagnostics and may cause CNS disorders, further highlighting their importance. This review focuses on the evolution and development of the brain ventricular system, associated function, and connected pathologies. It is geared as an introduction for scholars with little background in the field.

Absolute reliability and concurrent validity of a novel electromechanical pulley dynamometer for measuring shoulder rotation isometric strength in asymptomatic subjects. Study conducted at Pontificia Universidad Católica, Santiago, Chile.

OBJECTIVE: To estimate the test-retest reliability of measurements in shoulder internal and external rotators' isometric peak torque using a new dynamometer, and to compare it with isokinetic dynamometer. METHODS: The validity study was conducted in September-October 2016 at Pontificia Universidad Catolica de Chile and the Clinica Las Condes, Santiago, Chile. It comprised of asymptomatic university students who were randomly tested twice within a two-week period while in a supine position at 90° of shoulder abduction, using the novel functional electromechanical pulley dynamometer. Concurrent validity was assessed through comparing the values with the gold standard isokinetic dynamometer in the same position. SPSS 17 was used for data analysis. RESULTS: Of the 24 subjects, 5(21%) were males and 19(79%) were females. The overall mean age was 23.1±2.2 years, body mass index 23.6±2.13 kg/m2 and Shoulder Pain and Disability Index score was 3.9±6.4. There was no statistically non-significant difference in terms of test-retest trials and between the devices (p>0.05). Absolute reliability was 24.3% for internal rotation and 27.9% for external rotation. Both dynamometer systems were very highly correlated for internal rotators peak torque (0.93) and highly correlated for external rotators peak torque (0.84). CONCLUSIONS: Compared to the gold standard, the new device was found to be a valid instrument in measuring maximal voluntary isometric peak torque in internal and external rotation.

Molecular Identification, Antifungal Susceptibility, and Geographic Origin of Clinical Strains of Sporothrix schenckii Complex in Mexico.

Sporotrichosis is a subcutaneous mycosis caused by Sporothrix schenckii complex. The disease has been reported worldwide. However, the incidence of the etiological agent varies in its geographic distribution. We studied 39 clinical isolates of Sporothrix schenckii from diverse regions in Mexico, collected from 1998 to 2016. Molecular identification was performed by sequence analysis of the partial calmodulin gene. In vitro antifungal susceptibility to amphotericin B (AMB), itraconazole (ITC), voriconazole (VRC), posaconazole (PSC), fluconazole (FLC), terbinafine (TRB), caspofungin (CSF), anidulafungin (ANF), and micafungin (MCF) was evaluated. Thirty-eight isolates of S. schenckii complex were divided into five supported clades in a phylogenetic tree. The predominant clinical form was lymphocutaneous (92.3%), fixed cutaneous (5.1%), and disseminated (2.5%). Terbinafine exhibited the best in vitro antifungal activity, while fluconazole was ineffective against Sporothrix schenckii complex. Our results showed diverse geographic distribution of clinical isolates in eight states; definitive identification was done by CAL gen PCR-sequencing. In Mexico, S. schenckii is considered to be an etiological agent of human sporotrichosis cases, and lymphocutaneous is the most prevalent form of the disease. This study revealed four clades of S. schenckiisensu stricto by phylogenetic analysis. Furthermore, we report one case of S. globosa isolated from human origin from the North of Mexico.