Building synthetic chromosomes from natural DNA.

De novo chromosome synthesis is costly and time-consuming, limiting its use in research and biotechnology. Building synthetic chromosomes from natural components is an unexplored alternative with many potential applications. In this paper, we report CReATiNG (Cloning, Reprogramming, and Assembling Tiled Natural Genomic DNA), a method for constructing synthetic chromosomes from natural components in yeast. CReATiNG entails cloning segments of natural chromosomes and then programmably assembling them into synthetic chromosomes that can replace the native chromosomes in cells. We use CReATiNG to synthetically recombine chromosomes between strains and species, to modify chromosome structure, and to delete many linked, non-adjacent regions totaling 39% of a chromosome. The multiplex deletion experiment reveals that CReATiNG also enables recovery from flaws in synthetic chromosome design via recombination between a synthetic chromosome and its native counterpart. CReATiNG facilitates the application of chromosome synthesis to diverse biological problems.

Application and performance of heart-rate-based methods to estimate oxygen consumption at different exercise intensities in postmenopausal women.

PURPOSE: Heart rate (HR) is a widespread method to estimate oxygen consumption ([Formula: see text]O2), exercise intensity, volume, and energy expenditure. Still, accuracy depends on lab tests or using indexes like HRnet and HRindex. This study addresses HR indexes' applicability in postmenopausal women (PMW), who constitute over 50% of the aging population and may have unique characteristics (e.g., heart size) affecting HR use. METHODS: Fourteen PMW underwent a cycling ramp incremental test to establish the relationships between [Formula: see text]O2 (in MET) and absolute HR, HRnet, and HRindex. In a second group of ten PMW, population-specific and general equations were tested to predict MET and energy expenditure during six constant work exercises at various intensities. Pulmonary gas exchange and HR were continuously measured using a metabolic cart. Correlations, Bland-Altman analysis, and two-way RM-ANOVA were used to compare estimated and measured values. RESULTS: Strong linear relationships between the three HR indexes and MET were found in Group 1. In Group 2, population-specific equations showed medium-to-high correlations, precision, and no significant biases when estimating MET and energy expenditure. HRnet and HRindex outperformed absolute HR in accuracy. General HR equations had similar correlations but exhibited larger biases and imprecision. Statistical differences between measured and estimated values were observed at all intensities with general equations. CONCLUSION: This investigation confirms the suitability of HR for estimating aerobic metabolism in one of the most significant aging populations. However, it emphasizes the importance of considering individual variability and developing population-specific models when utilizing HR to infer metabolism.

Charge carrier transport in perylene-based and pyrene-based columnar liquid crystals.

Electron and hole transport characteristics were evaluated for perylene-based and pyrene-based compounds using electron-only and hole-only devices. The perylene presented a columnar hexagonal liquid crystal phase at room temperature with strong molecular π-stacking inside the columns. The pyrene crystallizes bellow 166 °C, preserving the close-packed columnar rectangular structure of the mesophase. Photophysical analysis and numerical calculations assisted the interpretation of positive and negative charge carrier mobilities obtained from fitting the space charge limited regime of current vs voltage curves. The pyrene-based material demonstrated an electron mobility two orders of magnitude higher than the perylene one, indicating the potential of this class of materials as electron transporting layer.

Effect of acute heat exposure on the determination of critical power and W' in women and men.

The goal of this study was to investigate to what extent acute heat exposure would affect the parameters of the power-duration relationship, i.e. CP and W', using multiple constant workload tests to task failure, in women and men. Twenty four young physically active participants (12 men, 12 women) performed 3-5 constant load tests to determine CP and W', both in temperate (TEMP; 18°C) and hot (HOT; 36°C) environmental conditions. A repeated-measures ANOVA was executed to find differences between TEMP and HOT, and between women and men. In HOT, CP was reduced by 6.5% (227 ± 50 vs. 212 ± 47 W), while W' increased 12.4% (16.4 ± 4.4 vs. 18.5 ± 5.6 kJ). No significant two-way sex × temperature interactions were observed, indicating that the environmental conditions did not have a different effect in men compared with women. The intersection of the average curvatures in TEMP and HOT occurred at 137 s and 280 W in women, and 153 s and 397 W in men. Acute heat exposure had an impact on the parameters CP and W', i.e. CP decreased whereas W' increased. The increase in W' might be a consequence of the mathematical modelling for the used test methodology, rather than a physiological accurate value of W' in HOT. No differences induced by heat exposure were observed between women and men.

The determination of CP and W' was done using multiple constant workload tests to task failure and acute heat exposure induced changes in CP (decrease) and W' (increase).The increase in W' with acute heat exposure might be a consequence of the mathematical modelling for the used test methodology, rather than a physiological accurate value of W'.Acute heat exposure had a similar effect on performance parameters in women and men.

Critical power, W' and W' reconstitution in women and men.

PURPOSE: The aim of this study was to compare critical power (CP) and work capacity W', and W' reconstitution (W'REC) following repeated maximal exercise between women and men. METHODS: Twelve women ([Formula: see text]O2PEAK: 2.53 ± 0.37 L·min-1) and 12 men ([Formula: see text]O2PEAK: 4.26 ± 0.30 L·min-1) performed a minimum of 3 constant workload tests, to determine CP and W', and 1 maximal exercise repetition test with three work bouts (WB) to failure, to quantify W'REC during 2 recovery periods, i.e., W'REC1 and W'REC2. An independent samples t test was used to compare CP and W' values between women and men, and a repeated-measures ANOVA was used to compare W'REC as fraction of W' expended during the first WB, absolute W'REC, and normalized to lean body mass (LBM). RESULTS: CP normalized to LBM was not different between women and men, respectively, 3.7 ± 0.5 vs. 4.1 ± 0.4 W·kgLBM-1, while W' normalized to LBM was lower in women 256 ± 29 vs. 305 ± 45 J·kgLBM-1. Fractional W'REC1 was higher in women than in men, respectively, 74.0 ± 12.0% vs. 56.8 ± 9.5%. Women reconstituted less W' than men in absolute terms (8.7 ± 1.2 vs. 10.9 ± 2.0 kJ) during W'REC1, while normalized to LBM no difference was observed between women and men (174 ± 23 vs. 167 ± 31 J·kgLBM-1). W'REC2 was lower than W'REC1 both in women and men. CONCLUSION: Sex differences in W'REC (absolute women < men; fractional women > men) are eliminated when LBM is accounted for. Prediction models of W'REC might benefit from including LBM as a biological variable in the equation. This study confirms the occurrence of a slowing of W'REC during repeated maximal exercise.

Building synthetic chromosomes from natural DNA.

De novo chromosome synthesis is costly and time-consuming, limiting its use in research and biotechnology. Building synthetic chromosomes from natural components is an unexplored alternative with many potential applications. In this paper, we report CReATiNG (Cloning, Reprogramming, and Assembling Tiled Natural Genomic DNA), a method for constructing synthetic chromosomes from natural components in yeast. CReATiNG entails cloning segments of natural chromosomes and then programmably assembling them into synthetic chromosomes that can replace the native chromosomes in cells. We used CReATiNG to synthetically recombine chromosomes between strains and species, to modify chromosome structure, and to delete many linked, non-adjacent regions totaling 39% of a chromosome. The multiplex deletion experiment revealed that CReATiNG also enables recovery from flaws in synthetic chromosome design via recombination between a synthetic chromosome and its native counterpart. CReATiNG facilitates the application of chromosome synthesis to diverse biological problems.

Cholinergic regulation of vascular endothelial function by human ChAT+ T cells.

Endothelial dysfunction and impaired vasodilation are linked with adverse cardiovascular events. T lymphocytes expressing choline acetyltransferase (ChAT), the enzyme catalyzing biosynthesis of the vasorelaxant acetylcholine (ACh), regulate vasodilation and are integral to the cholinergic antiinflammatory pathway in an inflammatory reflex in mice. Here, we found that human T cell ChAT mRNA expression was induced by T cell activation involving the PI3K signaling cascade. Mechanistically, we identified that ChAT mRNA expression was induced following the attenuation of RE-1 Silencing Transcription factor REST-mediated methylation of the ChAT promoter, and that ChAT mRNA expression levels were up-regulated by GATA3 in human T cells. In functional experiments, T cell-derived ACh increased endothelial nitric oxide-synthase activity, promoted vasorelaxation, and reduced vascular endothelial activation and promoted barrier integrity by a cholinergic mechanism. Further, we observed that survival in a cohort of patients with severe circulatory failure correlated with their relative frequency of ChAT +CD4+ T cells in blood. These findings on ChAT+ human T cells provide a mechanism for cholinergic immune regulation of vascular endothelial function in human inflammation.

Easy Prediction of the Maximal Lactate Steady-State in Young and Older Men and Women.

Maximal Lactate steady-state (MLSS) demarcates sustainable from unsustainable exercise and is used for evaluation/monitoring of exercise capacity. Still, its determination is physically challenging and time-consuming. This investigation aimed at validating a simple, submaximal approach based on blood lactate accumulation ([Δlactate]) at the third minute of cycling in a large cohort of men and women of different ages. 68 healthy adults (40♂, 28♀, 43 ± 17 years (range 19-78), VO2max 45 ± 11 ml-1·kg-1·min-1 (25-68)) performed 3-5 constant power output (PO) trials with a target duration of 30 minutes to determine the PO corresponding to MLSS. During each trial, [Δlactate] was calculated as the difference between the third minute and baseline. A multiple linear regression was computed to estimate MLSS based on [Δlactate], subjects` gender, age and the trial PO. The estimated MLSS was compared to the measured value by paired t-test, correlation, and Bland-Altman analysis. The group mean value of estimated MLSS was 180 ± 51 W, not significantly different from (p = 0.98) and highly correlated with (R2 = 0.89) measured MLSS (180 ± 54 watts). The bias between values was 0.17 watts, and imprecision 18.2 watts. This simple, submaximal, time- and cost-efficient test accurately and precisely predicts MLSS across different samples of healthy individuals (adjusted R2 = 0.88) and offers a practical and valid alternative to the traditional MLSS determination.

The effect of acute heat exposure on the determination of exercise thresholds from ramp and step incremental exercise.

PURPOSE: The aim of this study was to examine how respiratory (RT) and lactate thresholds (LT) are affected by acute heat exposure in the two most commonly used incremental exercise test protocols (RAMP and STEP) for functional evaluation of aerobic fitness, exercise prescription and monitoring training intensities. METHODS: Eleven physically active male participants performed four incremental exercise tests, two RAMP (30 W·min-1) and two STEP (40 W·3 min-1), both in 18 °C (TEMP) and 36 °C (HOT) with 40% relative humidity to determine 2 RT and 16 LT, respectively. Distinction was made within LT, taking into account the individual lactate kinetics (LTIND) and fixed value lactate concentrations (LTFIX). RESULTS: A decrease in mean power output (PO) was observed in HOT at LT (-6.2 ± 1.9%), more specific LTIND (-5.4 ± 1.4%) and LTFIX (-7.5 ± 2.4%), compared to TEMP, however not at RT (-1.0 ± 2.7%). The individual PO difference in HOT compared to TEMP over all threshold methods ranged from -53 W to +26 W. Mean heart rate (HR) did not differ in LT, while it was increased at RT in HOT (+10 ± 8 bpm). CONCLUSION: This study showed that exercise thresholds were affected when ambient air temperature was increased. However, a considerable degree of variability in the sensitivity of the different threshold concepts to acute heat exposure was found and a large individual variation was noticed. Test design and procedures should be taken into account when interpreting exercise test outcomes.

Physical Preparation of a World-Class Lightweight Men's Double Sculls Team for the Tokyo 2020 Olympics.

PURPOSE: To analyze the physical profile and training program of a world-class lightweight double sculls rowing crew toward the Tokyo 2020 Olympics. METHOD: A case study in which both rowers performed physical testing in November 2020 and April 2021 (anthropometrics, incremental rowing test, and power profiling). The training program (38 wk) in the buildup to the Olympics was analyzed, providing insight into training characteristics (volume; contribution of rowing, alternative, and strength training; prescribed and recorded [heart rate] training-intensity distribution). The entire period was split into 3 phases: preparation period (8 wk), competition period 1 (11 wk), and competition period 2 (9 wk), and training characteristics were compared. RESULTS: In the April 2021 testing, rower A (1.89 m, 74.6 kg, 4.4% body fat) had a peak oxygen uptake of 5.8 L·min-1 (77.8 mL·min-1·kg-1) and a peak power output of 491 W. Rower B (1.82 m, 70.6 kg, 7.8% body fat) had a peak oxygen uptake of 5.5 L·min-1 (77.9 mL·min-1·kg-1) and a peak power output of 482 W. The mean weekly training volume was 14 hours 47 minutes (4 h 5 min), of which 58.5% (14.6%) consisted of rowing, 13.4% (6.8%) strength training, and 28.1% (2.6%) alternative training. Heart-rate training-intensity distribution was 77.8% (4.2%) in zone 1, 16.6% (3.7%) in zone 2, and 5.6% (2.8%) in zone 3 with a lower contribution of zone 1 in competition period 1 (P = .029) and competition period 2 (P = .023) compared with the preparation period, and a higher contribution of zone 3 in competition period 1 (P = .018) and competition period 2 (P = .011) compared with the preparation period. CONCLUSION: The crew combined a high volume of rowing, alternative, and strength training in a pyramidal heart-rate training-intensity distribution throughout the year.

Desempeño físico en pacientes con esclerosis lateral amiotrófica y su relación con el uso de silla de ruedas y ayuda para la marcha / Physical performance in patients with amyotrophic lateral sclerosis and its relationship with wheelchairs and walking aids use

Introducción y objetivos: El déficit de marcha y las caídas en pacientes con esclerosis lateral amiotrófica (ELA) restringen la movilidad. El objetivo de este estudio es examinar el uso apropiado de dispositivos de ayuda para la marcha y silla de ruedas para deambular basados en el riesgo de caídas y la velocidad de marcha de pacientes con ELA. Métodos: Se incluyeron datos retrospectivos de pacientes de la clínica ELA. Se registraron edad, género, tiempo de evolución, uso de silla de ruedas, dispositivo de ayuda para la marcha, velocidad de marcha y escala de Berg. Se relacionaron las variables categóricas con la prueba de chi cuadrado y test exacto de Fisher. Resultados: Cincuenta y ocho pacientes cumplieron con los criterios de inclusión. Veintisiete (46,55%) tuvieron uso adecuado de silla de ruedas basado en la velocidad de marcha (p=0,50). Esta asociación disminuye al 6% en pacientes que caminaron a velocidad menor a 0,88m/s. Cuarenta y dos (70,69%) tuvieron uso adecuado de dispositivo de ayuda para la marcha basado en el riesgo de caídas (P=0,001). Del subgrupo con puntaje de Berg ≤ 45, el 38% no empleó el dispositivo apropiado (p=0,06). Conclusiones: Los pacientes con severo déficit de equilibrio y de marcha tuvieron un uso inapropiado de dispositivos para caminar y/o silla de ruedas. Los hallazgos del presente estudio pueden significar un aporte que podría ser considerado en las evaluaciones clínicas de pacientes con ELA para minimizar los riesgos y mejorar la participación de este colectivo de personas.

Introduction and objectives: Gait deficits and falls in patients with amyotrophic lateral sclerosis (ALS) restrict mobility. The aim of this study is to examine the appropriate use of walking aids and wheelchairs, based on the risk of falling and walking speed of patients with ALS. Methods: Retrospective data from patients from the ALS clinic were included. Age, gender, evolution time, wheelchair use, walking aids, gait speed and the Berg Balance Scale were registered. Categorical variables were related to the Chi Square test and Fisher's exact test. Results: Fifty eight patients met the inclusion criteria. Twenty-seven (46.55%) had adequate use of a wheelchair based on walking speed (p=.50). This association decreases to 6% in patients who walked at a speed lower than 0.88m/s. Forty-two (70.69%) had adequate use of an assistive device based on the risk of falls (p=.001). Of the subgroup with Berg Balance score ≤ 45, 38% did not use the appropriate device (p=.06). Conclusion: Patients with severe gait and balance deficits had inappropriate use of walking aids and/or wheelchairs. The findings of this study may mean a contribution that could be considered in the clinical evaluations of patients with ALS to minimize risks and improve the participation of this group of people.(AU)

Tracking the turnover of SARS-CoV-2 VOCs Gamma to Delta in a Brazilian state (Minas Gerais) with a high-vaccination status.

The emergence and global dissemination of Severe Acute Respiratory Syndrome virus 2 (SARS-CoV-2) variants of concern (VOCs) have been described as the main factor driving the Coronavirus Disease 2019 pandemic. In Brazil, the Gamma variant dominated the epidemiological scenario during the first period of 2021. Many Brazilian regions detected the Delta variant after its first description and documented its spread. To monitor the introduction and spread of VOC Delta, we performed Polymerase Chain Reaction (PCR) genotyping and genome sequencing in ten regional sentinel units from June to October 2021 in the State of Minas Gerais (MG). We documented the introduction and spread of Delta, comprising 70 per cent of the cases 8 weeks later. Comparing the viral loads of the Gamma and Delta dominance periods, we provide additional evidence that the latter is more transmissible. The spread and dominance of Delta did not culminate in the increase in cases and deaths, suggesting that the vaccination may have restrained the epidemic growth. Analysis of 224 novel Delta genomes revealed that Rio de Janeiro state was the primary source for disseminating this variant in the state of MG. We present the establishment of Delta, providing evidence of its enhanced transmissibility and showing that this variant shift did not aggravate the epidemiological scenario in a high immunity setting.

Major Regional-Scale Production of O3 and Secondary Organic Aerosol in Remote Amazon Regions from the Dynamics and Photochemistry of Urban and Forest Emissions.

The Amazon rainforest suffers increasing pressure from anthropogenic activities. A key aspect not fully understood is how anthropogenic atmospheric emissions within the basin interact with biogenic emissions and impact the forest's atmosphere and biosphere. We combine a high-resolution atmospheric chemical transport model with an improved emissions inventory and in-situ measurements to investigate a surprisingly high concentration of ozone (O3) and secondary organic aerosol (SOA) 150-200 km downwind of Manaus city in an otherwise pristine forested region. We show that atmospheric dynamics and photochemistry determine a gross production of secondary pollutants seen in the simulation. After sunrise, the erosion of the nocturnal boundary layer mixes natural forest emissions, rich in biogenic volatile organic compounds, with a lofted pollution layer transported overnight, rich in nitrogen oxides and formaldehyde. As a result, O3 and SOA concentrations greater than ∼47 ppbv and 1.8 µg m-3, respectively, were found, with maximum concentrations occurring at 2 pm LT, 150-200 km downwind of Manaus city. These high concentrations affect a large primary forested area of about 11,250 km2. These oxidative areas are under a NOx-limited regime so that changes in NOx emissions from Manaus have a significant impact on O3 and SOA production.

Patterns of disease relapse and posttreatment follow-up of patients with resected pancreatic adenocarcinoma: A single-center analysis.

BACKGROUND AND OBJECTIVES: To describe the patterns of disease relapse and follow-up of patients with resected pancreatic adenocarcinoma. Additionally, we looked at patients' characteristics at relapse and survival. METHODS: We included patients with potentially resectable pancreatic adenocarcinoma diagnosed from 2008 to 2018 who were submitted to resection with clear macroscopic margins and started posttreatment surveillance. RESULTS: The study population consists of 73 patients. The median interval between imaging studies was 3.2 months during the first 2 years of follow-up and 5.1 months thereafter. Forty-eight patients (65.8%) experienced disease relapse. The most frequent single site of relapse was locoregional (N = 21; 43.8%). At relapse, 31 patients (64.6%) were symptomatic and forty-two patients (87.6%) had Eastern Cooperative Oncology Group performance status 0 or 1. Most patients were able to undergo additional anticancer therapy (N = 41; 85.4%). Patients with asymptomatic relapses experienced longer median postrelapse survival (25.4 vs. 11.3 months; p = 0.015). CONCLUSIONS: A follow-up protocol that included imaging studies every 3 months in the first 2 years and every 6 months thereafter is able to diagnose disease relapse when patients have adequate performance status and are still able to undergo additional anticancer treatment.

Brazilian Society of Surgical Oncology surgical standards for resectable colorectal cancer liver metastases.

The management of colorectal cancer liver metastasis (CRLM) has become complex because of the increasing availability of medical, radiological, and surgical treatment options applied either alone or in combination. However, resection remains the only evidence-based curative therapy. These Brazilian Society of Surgical Oncology surgical standards are intended to guide clinicians in the decision-making process for modern surgical management of CRLM within a multidisciplinary team in an evidence-based framework, focusing on resectable disease.

Surgery or active surveillance for pNETs < 2 cm: Preliminary results from a single center Brazilian cohort.

BACKGROUND AND OBJECTIVES: Incidence of pancreatic neuroendocrine tumors (pNETS) seems to be rising over the years, with many cases incidentally diagnosed. Surgery and active surveillance are current treatment modalities for small pNETS. We review our institutional series and compare outcomes for small asymptomatic and nonfunctioning tumors. METHODS: This retrospective cohort study included patients with 2 cm or less and well differentiated pNETS at a single Brazilian Cancer Center. From 2002 to 2020, patients received active surveillance or surgery as a treatment strategy. Short and long-term results were compared. RESULTS: Sixty-four patients were included, 41 in surgical strategy and 23 in the active surveillance approach. Baseline group characteristics were comparable. More patients on active surveillance underwent abdominal magnetic resonance imaging (MRI) and had tumors located in the pancreatic head (41% vs. 17%, p = 0.038). Minimally invasive procedure was chosen in 80.1% of the surgical patients. No patient died after surgery. Median follow-up period was 38.6 and 46.4 months for active surveillance and surgery cohorts, respectively. No difference in disease progression rate was observed. CONCLUSION: Both approaches seem to be safe for small pNETs. Long-term outcome and quality of life should be considered when discussing such options with patients.

PD-L1 expression in gastric and gastroesophageal junction cancer patients treated with perioperative chemotherapy.

BACKGROUND AND OBJECTIVES: The incidence, predictive, and prognostic impact of programmed cell death (PD-L1) expression in gastric (GC) and gastroesophageal junction tumors (GEJC) treated with perioperative chemotherapy is poorly understood. We aimed to assess PD-L1 expression by immunohistochemistry (IHC) in both pre and posttreatment specimens evaluating its impact on pathological response and survival outcomes. METHODS: Retrospective cohort of patients with GC and GEJ tumors treated in a single western cancer center between 2007 and 2017. PD-L1 expression was assessed by IHC before and after neoadjuvant chemotherapy, in surgical samples, and reported as combined positive score (CPS). CPS > 1% was tested for its association with pathological response and overall survival (OS). RESULTS: We were able to assess PD-L1 expression in at least one tissue sample from 155 subjects. PD-L1 positivity rate was 20%. In 74 paired samples, a 21% discordance between PD-L1 expression in biopsy sample and surgical specimen was observed. With a median follow-up period of 60.3 months, 5-years disease-free survival was 60.5% with a median OS not reached. PD-L1 expression was neither associated with pathological response or survival outcomes. CONCLUSIONS: PD-L1 expression in the setting of locally advanced GC tumors was relatively low and can vary considering the tissue sample analyzed. This expression had no association with survival or pathological response in this population.

Noninvasive Stimulation of Peripheral Nerves using Temporally-Interfering Electrical Fields.

Electrical stimulation of peripheral nerves is a cornerstone of bioelectronic medicine. Effective ways to accomplish peripheral nerve stimulation (PNS) noninvasively without surgically implanted devices are enabling for fundamental research and clinical translation. Here, it is demonstrated how relatively high-frequency sine-wave carriers (3 kHz) emitted by two pairs of cutaneous electrodes can temporally interfere at deep peripheral nerve targets. The effective stimulation frequency is equal to the offset frequency (0.5 - 4 Hz) between the two carriers. This principle of temporal interference nerve stimulation (TINS) in vivo using the murine sciatic nerve model is validated. Effective actuation is delivered at significantly lower current amplitudes than standard transcutaneous electrical stimulation. Further, how flexible and conformable on-skin multielectrode arrays can facilitate precise alignment of TINS onto a nerve is demonstrated. This method is simple, relying on the repurposing of existing clinically-approved hardware. TINS opens the possibility of precise noninvasive stimulation with depth and efficiency previously impossible with transcutaneous techniques.