Flexible structural arrangement and DNA-binding properties of protein p6 from Bacillus subtillis phage φ29.

The genome-organizing protein p6 of Bacillus subtilis bacteriophage φ29 plays an essential role in viral development by activating the initiation of DNA replication and participating in the early-to-late transcriptional switch. These activities require the formation of a nucleoprotein complex in which the DNA adopts a right-handed superhelix wrapping around a multimeric p6 scaffold, restraining positive supercoiling and compacting the viral genome. Due to the absence of homologous structures, prior attempts to unveil p6's structural architecture failed. Here, we employed AlphaFold2 to engineer rational p6 constructs yielding crystals for three-dimensional structure determination. Our findings reveal a novel fold adopted by p6 that sheds light on its self-association mechanism and its interaction with DNA. By means of protein-DNA docking and molecular dynamic simulations, we have generated a comprehensive structural model for the nucleoprotein complex that consistently aligns with its established biochemical and thermodynamic parameters. Besides, through analytical ultracentrifugation, we have confirmed the hydrodynamic properties of the nucleocomplex, further validating in solution our proposed model. Importantly, the disclosed structure not only provides a highly accurate explanation for previously experimental data accumulated over decades, but also enhances our holistic understanding of the structural and functional attributes of protein p6 during φ29 infection.

Molecular architecture and oligomerization of Candida glabrata Cdc13 underpin its telomeric DNA-binding and unfolding activity.

The CST complex is a key player in telomere replication and stability, which in yeast comprises Cdc13, Stn1 and Ten1. While Stn1 and Ten1 are very well conserved across species, Cdc13 does not resemble its mammalian counterpart CTC1 either in sequence or domain organization, and Cdc13 but not CTC1 displays functions independently of the rest of CST. Whereas the structures of human CTC1 and CST have been determined, the molecular organization of Cdc13 remains poorly understood. Here, we dissect the molecular architecture of Candida glabrata Cdc13 and show how it regulates binding to telomeric sequences. Cdc13 forms dimers through the interaction between OB-fold 2 (OB2) domains. Dimerization stimulates binding of OB3 to telomeric sequences, resulting in the unfolding of ssDNA secondary structure. Once bound to DNA, Cdc13 prevents the refolding of ssDNA by mechanisms involving all domains. OB1 also oligomerizes, inducing higher-order complexes of Cdc13 in vitro. OB1 truncation disrupts these complexes, affects ssDNA unfolding and reduces telomere length in C. glabrata. Together, our results reveal the molecular organization of C. glabrata Cdc13 and how this regulates the binding and the structure of DNA, and suggest that yeast species evolved distinct architectures of Cdc13 that share some common principles.

A bout of aerobic exercise in the heat increases carbohydrate use but does not enhance the disposal of an oral glucose load, in healthy active individuals.

We investigated if a bout of exercise in a hot environment (HEAT) would reduce the postprandial hyperglycemia induced by glucose ingestion. The hypothesis was that HEAT stimulating carbohydrate oxidation and glycogen use would increase the disposal of an ingested glucose load [i.e., oral glucose tolerance test (OGTT); 75 g of glucose]. Separated by at least 1 wk, nine young healthy individuals underwent three trials after an overnight fast in a randomized order. Two trials included 50 min of pedaling at 58 ± 5% V̇o2max either in a thermoneutral (21 ± 1°C; NEUTRAL) or in a hot environment (33 ± 1°C; HEAT) eliciting similar energy expenditure (503 ± 101 kcal). These two trials were compared with a no-exercise trial (NO EXER). Twenty minutes after exercise (or rest), subjects underwent an OGTT, while carbohydrate oxidation (CHOxid, using indirect calorimetry) plasma blood glucose, insulin concentrations (i.e., [glucose], [insulin]), and double tracer glucose kinetics ([U-13C] glucose ingestion and [6,6-2H2] glucose infusion) were monitored for 120 min. At rest, [glucose], [insulin], and rates of appearance/disappearance of glucose in plasma (glucose Ra/Rd) were similar among trials. During exercise, heart rate, tympanic temperature, [glucose], glycogen oxidation, and total CHOxid were higher during HEAT than NEUTRAL (i.e., 149 ± 35 vs. 124 ± 31 µmol·kg-1·min-1, P = 0.010). However, during the following OGTT, glucose Rd was similar in HEAT and NEUTRAL trials (i.e., 25.1 ± 3.6 vs. 25.2 ± 5.3 µmol·kg-1·min-1, P = 0.981). Insulin sensitivity (i.e., ISIndexMATSUDA) only improved in NEUTRAL compared with NO EXER (10.1 ± 4.6 vs. 8.8 ± 3.7 au; P = 0.044). In summary, stimulating carbohydrate use with exercise in a hot environment does not improve postprandial plasma glucose disposal or insulin sensitivity in a subsequent OGTT.NEW & NOTEWORTHY Exercise in the heat increases estimated muscle glycogen use. Reduced muscle glycogen after exercise in the heat could increase insulin-mediated glucose uptake during a subsequent oral glucose tolerance test (OGTT). However, plasma glucose kinetics are not improved during the OGTT in response to a bout of exercise in the heat, and insulin sensitivity worsens. Heat stress activates glucose counterregulatory hormones whose actions may linger during the OGTT, preventing increased glucose uptake.

The interaction of the ribotoxin α-sarcin with complex model lipid vesicles.

Fungal ribotoxins are extracellular RNases that inactivate ribosomes by cleaving a single phosphodiester bond at the universally conserved sarcin-ricin loop of the large rRNA. However, to reach the ribosomes, they need to cross the plasma membrane. It is there where these toxins show their cellular specificity, being especially active against tumoral or virus-infected cells. Previous studies have shown that fungal ribotoxins interact with negatively charged membranes, typically containing phosphatidylserine or phosphatidylglycerol. This ability is rooted on their long, non-structured, positively charged loops, and its N-terminal ß-hairpin. However, its effect on complex lipid mixtures, including sphingophospholipids or cholesterol, remains poorly studied. Here, wild-type α-sarcin was used to evaluate its interaction with a variety of membranes not assayed before, which resemble much more closely mammalian cell membranes. The results confirm that α-sarcin is particularly sensitive to charge density on the vesicle surface. Its ability to induce vesicle aggregation is strongly influenced by both the lipid headgroup and the degree of saturation of the fatty acid chains. Acyl chain length is indeed particularly important for lipid mixing. Finally, cholesterol plays an important role in diluting the concentration of available negative charges and modulates the ability of α-sarcin to cross the membrane.

Glucose volume of distribution affects insulin sensitivity measured by intravenous glucose tolerance test.

AIM: To determine whether glucose volume of distribution (VdGLUCOSE ) affects the diagnosis of impaired insulin sensitivity (IS) when using an intravenous glucose tolerance test (IVGTT). METHODS: Individuals with distinct levels of IS underwent IVGTT after an overnight fast. The prediabetic group (Prediab; n = 33) differed from the healthy group (Healthy; n = 14) in their larger glycosylated hemoglobin (HbA1c of 5.9 ± 0.3 vs. 5.4 ± 0.1%; 41 ± 4 vs. 36 ± 1 mmol/mol; p < 0.001), percent body fat (37 ± 6 vs. 24 ± 3%; p < 0.001) and cardiovascular fitness level (VO2MAX 22 ± 5 vs. 44 ± 5 mL of O2 ·kg-1 ·min-1 ; p < 0.001). Ten minutes after intravenous infusion of the glucose bolus (i.e., 35 g in a 30% solution), VdGLUCOSE was assessed from the increases in plasma glucose concentration. IS was calculated during the next 50 min using the slope of glucose disappearance and the insulin time-response curve. RESULTS: VdGLUCOSE was higher in Healthy than in Prediab (230 ± 49 vs. 185 ± 21 mL·kg-1 ; p < 0.001). VdGLUCOSE was a strong predictor of IS (ß standardized coefficient 0.362; p = 0.004). VO2MAX was associated with VdGLUCOSE and IS (Pearson r = 0.582 and 0.704, respectively; p < 0.001). However, body fat was inversely associated with VdGLUCOSE and IS (r = -0.548 and -0.555, respectively; p < 0.001). CONCLUSIONS: Since fat mass is inversely related to VdGLUCOSE and in turn, VdGLUCOSE affects the calculations of IS, the IV glucose bolus dose should be calculated based on fat-free mass rather than body weight for a more accurate diagnosis of impaired IS.

Studying Macromolecular Interactions of Cellular Machines by the Combined Use of Analytical Ultracentrifugation, Light Scattering, and Fluorescence Spectroscopy Methods.

Cellular machines formed by the interaction and assembly of macromolecules are essential in many processes of the living cell. These assemblies involve homo- and hetero-associations, including protein-protein, protein-DNA, protein-RNA, and protein-polysaccharide associations, most of which are reversible. This chapter describes the use of analytical ultracentrifugation, light scattering, and fluorescence-based methods, well-established biophysical techniques, to characterize interactions leading to the formation of macromolecular complexes and their modulation in response to specific or unspecific factors. We also illustrate, with several examples taken from studies on bacterial processes, the advantages of the combined use of subsets of these techniques as orthogonal analytical methods to analyze protein oligomerization and polymerization, interactions with ligands, hetero-associations involving membrane proteins, and protein-nucleic acid complexes.

Assisted assembly of bacteriophage T7 core components for genome translocation across the bacterial envelope.

In most bacteriophages, genome transport across bacterial envelopes is carried out by the tail machinery. In viruses of the Podoviridae family, in which the tail is not long enough to traverse the bacterial wall, it has been postulated that viral core proteins assembled inside the viral head are translocated and reassembled into a tube within the periplasm that extends the tail channel. Bacteriophage T7 infects Escherichia coli, and despite extensive studies, the precise mechanism by which its genome is translocated remains unknown. Using cryo-electron microscopy, we have resolved the structure of two different assemblies of the T7 DNA translocation complex composed of the core proteins gp15 and gp16. Gp15 alone forms a partially folded hexamer, which is further assembled upon interaction with gp16 into a tubular structure, forming a channel that could allow DNA passage. The structure of the gp15-gp16 complex also shows the location within gp16 of a canonical transglycosylase motif involved in the degradation of the bacterial peptidoglycan layer. This complex docks well in the tail extension structure found in the periplasm of T7-infected bacteria and matches the sixfold symmetry of the phage tail. In such cases, gp15 and gp16 that are initially present in the T7 capsid eightfold-symmetric core would change their oligomeric state upon reassembly in the periplasm. Altogether, these results allow us to propose a model for the assembly of the core translocation complex in the periplasm, which furthers understanding of the molecular mechanism involved in the release of T7 viral DNA into the bacterial cytoplasm.

Efficacy of morning versus afternoon aerobic exercise training on reducing metabolic syndrome components: A randomized controlled trial.

A supervised intense aerobic exercise program improves the health of individuals with metabolic syndrome (MetS). However, it is unclear whether the timing of training within the 24 h day would influence those health benefits. The present study aimed to determine the influence of morning vs. afternoon exercise on body composition, cardiometabolic health and components of MetS. One hundred thirty-nine individuals with MetS were block randomized into morning (AMEX; n = 42) or afternoon (PMEX; n = 59) exercise training groups, or a non-training control group (Control; n = 38). Exercise training was comprised of 48 supervised high-intensity interval sessions distributed over 16 weeks. Body composition, cardiorespiratory fitness (assessed by V ̇ O 2 max ${\dot V_{{{\mathrm{O}}_{\mathrm{2}}}{\mathrm{max}}}}$ ), maximal fat oxidation (FOmax ), blood pressure and blood metabolites were assessed before and after the intervention. Compared with the non-training Control, both exercise groups improved similarly body composition (-0.7% fat loss; P = 0.002), waist circumference (-2.1 cm; P < 0.001), diastolic blood pressure (-3.8 mmHg; P = 0.004) and V ̇ O 2 max ${\dot V_{{{\mathrm{O}}_{\mathrm{2}}}{\mathrm{max}}}}$ (3.5 mL kg-1   min-1 ; P < 0.001) with no differences between training groups. AMEX, in comparison with PMEX, reduced systolic blood pressure (-4% vs. -1%; P = 0.019), plasma fasting insulin concentration (-12% vs. -5%; P = 0.001) and insulin resistance (-14% vs. -4%; P = 0.006). Furthermore, MetS Z score was further reduced in the AMEX compared to PMEX (-52% vs. -19%; P = 0.021) after training. In summary, high-intensity aerobic exercise training in the morning in comparison to training in the afternoon is somewhat more efficient at reducing cardiometabolic risk factors (i.e. systolic blood pressure and insulin sensitivity). KEY POINTS: The effect of exercise time of day on health promotion is an area that has gained interest in recent years; however, large-scale, randomized-control studies are scarce. People with metabolic syndrome (MetS) are at risk of developing cardiometabolic diseases and reductions in this risk with exercise training can be precisely gauged using a compound score sensitive to subtle evolution in each MetS component (i.e. Z score). Supervised aerobic exercise for 16 weeks (morning and afternoon), without dietary restriction, improved cardiorespiratory and metabolic fitness, body composition and mean arterial pressure compared to a non-exercise control group. However, training in the morning, without changes in exercise dose or intensity, reduced systolic blood pressure and insulin resistance further compared to when training in the afternoon. Thus, high-intensity aerobic exercise training in the morning is somewhat more efficient in improving the health of individuals with metabolic syndrome.

Metformin and exercise effects on postprandial insulin sensitivity and glucose kinetics in pre-diabetic and diabetic adults.

The potential interaction between metformin and exercise on glucose-lowering effects remains controversial. We studied the separated and combined effects of metformin and/or exercise on fasting and postprandial insulin sensitivity in individuals with pre-diabetes and type 2 diabetes (T2D). Eight T2D adults (60 ± 4 yr) with overweight/obesity (32 ± 4 kg·m-2) under chronic metformin treatment (9 ± 6 yr; 1281 ± 524 mg·day-1) underwent four trials; 1) taking their habitual metformin treatment (MET), 2) substituting during 96 h their metformin medication by placebo (PLAC), 3) placebo combined with 50 min bout of high-intensity interval exercise (PLAC + EX), and 4) metformin combined with exercise (MET + EX). Plasma glucose kinetics using stable isotopes (6,6-2H2 and [U-13C] glucose), and glucose oxidation by indirect calorimetry, were assessed at rest, during exercise, and in a subsequent oral glucose tolerance test (OGTT). Postprandial glucose and insulin concentrations were analyzed as mean and incremental area under the curve (iAUC), and insulin sensitivity was calculated (i.e., MATSUDAindex and OGISindex). During OGTT, metformin reduced glucose iAUC (i.e., MET and MET + EX lower than PLAC and PLAC + EX, respectively; P = 0.023). MET + EX increased MATSUDAindex above PLAC (4.8 ± 1.4 vs. 3.3 ± 1.0, respectively; P = 0.018) and OGISindex above PLAC (358 ± 52 vs. 306 ± 46 mL·min-1·m-2, respectively; P = 0.006). Metformin decreased the plasma appearance of the ingested glucose (Ra OGTT; MET vs. PLAC, -3.5; 95% CI -0.1 to -6.8 µmol·kg-1·min-1; P = 0.043). Metformin combined with exercise potentiates insulin sensitivity during an OGTT in individuals with pre-diabetes and type 2 diabetes. Metformin's blood glucose-lowering effect seems mediated by decreased oral glucose entering the circulation (gut-liver effect) an effect partially blunted after exercise.NEW & NOTEWORTHY Metformin is the most prescribed oral antidiabetic medicine in the world but its mechanism of action and its interactions with exercise are not fully understood. Our stable isotope tracer data suggested that metformin reduces the rates of oral glucose entering the circulation (gut-liver effect). Exercise, in turn, tended to reduce postprandial insulin blood levels potentiating metformin improvements in insulin sensitivity. Thus, exercise potentiates metformin improvements in glycemic control and should be advised to metformin users.

Sticholysin recognition of ceramide-phosphoethanolamine.

Actinoporins are pore-forming toxins produced by sea anemones. They exert their activity by binding to the membranes of target cells. There, they oligomerize, forming cation-selective pores, and inducing cell death by osmotic shock. In the early days of the field, it was shown that accessible sphingomyelin (SM) in the bilayer is required for the activity of actinoporins. While these toxins can also act on membranes composed solely of phosphatidylcholine (PC) with a high amount of cholesterol (Chol), consensus is that SM acts as a lipid receptor for actinoporins. It has been shown that the 2NH and 3OH moieties of SM are essential for actinoporin recognition. Hence, we wondered if ceramide-phosphoethanolamine (CPE) could also be recognized. Like SM, CPE has the 2NH and 3OH groups, and a positively charged headgroup. While actinoporins have been observed to affect membranes containing CPE, Chol was always also present, with the recognition of CPE remaining unclear. To test this possibility, we used sticholysins, produced by the Caribbean Sea anemone Stichodactyla helianthus. Our results show that sticholysins can induce calcein release on vesicles composed only of PC and CPE, in absence of Chol, in a way that is comparable to that induced on PC:SM membranes.

Crk proteins activate the Rap1 guanine nucleotide exchange factor C3G by segregated adaptor-dependent and -independent mechanisms.

BACKGROUND: C3G is a guanine nucleotide exchange factor (GEF) that activates Rap1 to promote cell adhesion. Resting C3G is autoinhibited and the GEF activity is released by stimuli that signal through tyrosine kinases. C3G is activated by tyrosine phosphorylation and interaction with Crk adaptor proteins, whose expression is elevated in multiple human cancers. However, the molecular details of C3G activation and the interplay between phosphorylation and Crk interaction are poorly understood. METHODS: We combined biochemical, biophysical, and cell biology approaches to elucidate the mechanisms of C3G activation. Binding of Crk adaptor proteins to four proline-rich motifs (P1 to P4) in C3G was characterized in vitro using isothermal titration calorimetry and sedimentation velocity, and in Jurkat and HEK293T cells by affinity pull-down assays. The nucleotide exchange activity of C3G over Rap1 was measured using nucleotide-dissociation kinetic assays. Jurkat cells were also used to analyze C3G translocation to the plasma membrane and the C3G-dependent activation of Rap1 upon ligation of T cell receptors. RESULTS: CrkL interacts through its SH3N domain with sites P1 and P2 of inactive C3G in vitro and in Jurkat and HEK293T cells, and these sites are necessary to recruit C3G to the plasma membrane. However, direct stimulation of the GEF activity requires binding of Crk proteins to the P3 and P4 sites. P3 is occluded in resting C3G and is essential for activation, while P4 contributes secondarily towards complete stimulation. Tyrosine phosphorylation of C3G alone causes marginal activation. Instead, phosphorylation primes C3G lowering the concentration of Crk proteins required for activation and increasing the maximum activity. Unexpectedly, optimal activation also requires the interaction of CrkL-SH2 domain with phosphorylated C3G. CONCLUSION: Our study revealed that phosphorylation of C3G by Src and Crk-binding form a two-factor mechanism that ensures tight control of C3G activation. Additionally, the simultaneous SH2 and SH3N interaction of CrkL with C3G, required for the activation, reveals a novel adaptor-independent function of Crk proteins relevant to understanding their role in physiological signaling and their deregulation in diseases. Video abstract.

Usefulness of a novel computer-aided detection system for colorectal neoplasia: a randomized controlled trial.

BACKGROUND AND AIMS: Artificial intelligence-based computer-aid detection (CADe) devices have been recently tested in colonoscopies, increasing the adenoma detection rate (ADR), mainly in Asian populations. However, evidence for the benefit of these devices in the occidental population is still low. We tested a new CADe device, namely, ENDO-AID (OIP-1) (Olympus, Tokyo, Japan), in clinical practice. METHODS: This randomized controlled trial included 370 consecutive patients who were randomized 1:1 to CADe (n = 185) versus standard exploration (n = 185) from November 2021 to January 2022. The primary endpoint was the ADR. Advanced adenoma was defined as ≥10 mm, harboring high-grade dysplasia, or with a villous pattern. Otherwise, the adenoma was nonadvanced. ADR was assessed in both groups stratified by endoscopist ADR and colon cleansing. RESULTS: In the intention-to-treat analysis, the ADR was 55.1% (102/185) in the CADe group and 43.8% (81/185) in the control group (P = .029). Nonadvanced ADRs (54.8% vs 40.8%, P = .01) and flat ADRs (39.4 vs 24.8, P = .006), polyp detection rate (67.1% vs 51%; P = .004), and number of adenomas per colonoscopy were significantly higher in the CADe group than in the control group (median [25th-75th percentile], 1 [0-2] vs 0 [0-1.5], respectively; P = .014). No significant differences were found in serrated ADR. After stratification by endoscopist and bowel cleansing, no statistically significant differences in ADR were found. CONCLUSIONS: Colonoscopy assisted by ENDO-AID (OIP-1) increases ADR and number of adenomas per colonoscopy, suggesting it may aid in the detection of colorectal neoplastic lesions, especially because of its detection of diminutive and flat adenomas. (Clinical trial registration number: NCT04945044.).

Chronic Statin Treatment Does Not Impair Exercise Lipolysis or Fat Oxidation in Exercise-Trained Individuals With Obesity and Dyslipidemia.

OBJECTIVE: To determine whether statin medication in individuals with obesity, dyslipidemia, and metabolic syndrome affects their capacity to mobilize and oxidize fat during exercise. METHODS: Twelve individuals with metabolic syndrome pedaled during 75 min at 54 ± 13% V˙O2max (5.7 ± 0.5 metabolic equivalents) while taking statins (STATs) or after 96-hr statin withdrawal (PLAC) in a randomized double-blind fashion. RESULTS: At rest, PLAC increased low-density lipoprotein cholesterol (i.e., STAT 2.55 ± 0.96 vs. PLAC 3.16 ± 0.76 mmol/L; p = .004) and total cholesterol blood levels (i.e., STAT 4.39 ± 1.16 vs. PLAC 4.98 ± 0.97 mmol/L; p = .008). At rest, fat oxidation (0.99 ± 0.34 vs. 0.76 ± 0.37 µmol·kg-1·min-1 for STAT vs. PLAC; p = .068) and the rates of plasma appearance of glucose and glycerol (i.e., Ra glucose-glycerol) were not affected by PLAC. After 70 min of exercise, fat oxidation was similar between trials (2.94 ± 1.56 vs. 3.06 ± 1.94 µmol·kg-1·min-1, STA vs. PLAC; p = .875). PLAC did not alter the rates of disappearance of glucose in plasma during exercise (i.e., 23.9 ± 6.9 vs. 24.5 ± 8.2 µmol·kg-1·min-1 for STAT vs. PLAC; p = .611) or the rate of plasma appearance of glycerol (i.e., 8.5 ± 1.9 vs. 7.9 ± 1.8 µmol·kg-1·min-1 for STAT vs. PLAC; p = .262). CONCLUSIONS: In patients with obesity, dyslipidemia, and metabolic syndrome, statins do not compromise their ability to mobilize and oxidize fat at rest or during prolonged, moderately intense exercise (i.e., equivalent to brisk walking). In these patients, the combination of statins and exercise could help to better manage their dyslipidemia.

Assessing the Feasibility of Replacing Subjective Questionnaire-Based Sleep Measurement with an Objective Approach Using a Smartwatch.

In order to ensure sufficient recovery of the human body and brain, healthy sleep is indispensable. For this purpose, appropriate therapy should be initiated at an early stage in the case of sleep disorders. For some sleep disorders (e.g., insomnia), a sleep diary is essential for diagnosis and therapy monitoring. However, subjective measurement with a sleep diary has several disadvantages, requiring regular action from the user and leading to decreased comfort and potential data loss. To automate sleep monitoring and increase user comfort, one could consider replacing a sleep diary with an automatic measurement, such as a smartwatch, which would not disturb sleep. To obtain accurate results on the evaluation of the possibility of such a replacement, a field study was conducted with a total of 166 overnight recordings, followed by an analysis of the results. In this evaluation, objective sleep measurement with a Samsung Galaxy Watch 4 was compared to a subjective approach with a sleep diary, which is a standard method in sleep medicine. The focus was on comparing four relevant sleep characteristics: falling asleep time, waking up time, total sleep time (TST), and sleep efficiency (SE). After evaluating the results, it was concluded that a smartwatch could replace subjective measurement to determine falling asleep and waking up time, considering some level of inaccuracy. In the case of SE, substitution was also proved to be possible. However, some individual recordings showed a higher discrepancy in results between the two approaches. For its part, the evaluation of the TST measurement currently does not allow us to recommend substituting the measurement method for this sleep parameter. The appropriateness of replacing sleep diary measurement with a smartwatch depends on the acceptable levels of discrepancy. We propose four levels of similarity of results, defining ranges of absolute differences between objective and subjective measurements. By considering the values in the provided table and knowing the required accuracy, it is possible to determine the suitability of substitution in each individual case. The introduction of a "similarity level" parameter increases the adaptability and reusability of study findings in individual practical cases.

Validating Force Sensitive Resistor Strip Sensors for Cardiorespiratory Measurement during Sleep: A Preliminary Study.

Sleep disorders can impact daily life, affecting physical, emotional, and cognitive well-being. Due to the time-consuming, highly obtrusive, and expensive nature of using the standard approaches such as polysomnography, it is of great interest to develop a noninvasive and unobtrusive in-home sleep monitoring system that can reliably and accurately measure cardiorespiratory parameters while causing minimal discomfort to the user's sleep. We developed a low-cost Out of Center Sleep Testing (OCST) system with low complexity to measure cardiorespiratory parameters. We tested and validated two force-sensitive resistor strip sensors under the bed mattress covering the thoracic and abdominal regions. Twenty subjects were recruited, including 12 males and 8 females. The ballistocardiogram signal was processed using the 4th smooth level of the discrete wavelet transform and the 2nd order of the Butterworth bandpass filter to measure the heart rate and respiration rate, respectively. We reached a total error (concerning the reference sensors) of 3.24 beats per minute and 2.32 rates for heart rate and respiration rate, respectively. For males and females, heart rate errors were 3.47 and 2.68, and respiration rate errors were 2.32 and 2.33, respectively. We developed and verified the reliability and applicability of the system. It showed a minor dependency on sleeping positions, one of the major cumbersome sleep measurements. We identified the sensor under the thoracic region as the optimal configuration for cardiorespiratory measurement. Although testing the system with healthy subjects and regular patterns of cardiorespiratory parameters showed promising results, further investigation is required with the bandwidth frequency and validation of the system with larger groups of subjects, including patients.

The Design and Development of Instrumented Toys for the Assessment of Infant Cognitive Flexibility.

The first years of an infant's life represent a sensitive period for neurodevelopment where one can see the emergence of nascent forms of executive function (EF), which are required to support complex cognition. Few tests exist for measuring EF during infancy, and the available tests require painstaking manual coding of infant behaviour. In modern clinical and research practice, human coders collect data on EF performance by manually labelling video recordings of infant behaviour during toy or social interaction. Besides being extremely time-consuming, video annotation is known to be rater-dependent and subjective. To address these issues, starting from existing cognitive flexibility research protocols, we developed a set of instrumented toys to serve as a new type of task instrumentation and data collection tool suitable for infant use. A commercially available device comprising a barometer and an inertial measurement unit (IMU) embedded in a 3D-printed lattice structure was used to detect when and how the infant interacts with the toy. The data collected using the instrumented toys provided a rich dataset that described the sequence of toy interaction and individual toy interaction patterns, from which EF-relevant aspects of infant cognition can be inferred. Such a tool could provide an objective, reliable, and scalable method of collecting early developmental data in socially interactive contexts.

Combined or Sequential Treatment with Immune Checkpoint Inhibitors and Car-T Cell Therapies for the Management of Haematological Malignancies: A Systematic Review.

The aim of this paper was to review the available evidence on the efficacy and safety of combined or sequential use of PD-1/PD-L1 immune checkpoint inhibitors (ICI) and CAR-T cell therapies in relapsed/refractory (R/R) haematological malignancies. A systematic literature review was performed until 21 November 2022. Inclusion criteria: cohort studies/clinical trials aimed at evaluating the efficacy and/or safety of the combination of CAR-T cell therapy with PD-1/PD-L1 inhibitors in R/R haematological malignancies, which had reported results. Those focusing only on ICI or CAR-T separately or evaluating the combination in other non-hematological solid tumours were excluded. We used a specific checklist for quality assessment of the studies, and then we extracted data on efficacy or efficiency and safety. A total of 1867 articles were identified, and 9 articles were finally included (early phase studies, with small samples of patients and acceptable quality). The main pathologies were B-cell acute lymphoblastic leukaemia (B-ALL) and B-cell non-Hodgkin's lymphoma (B-NHL). The most studied combination was tisagenlecleucel with pembrolizumab. In terms of efficacy, there is great variability: the combination could be a promising option in B-ALL, with modest data, and in B-NHL, although hopeful responses were received, the combination does not appear better than CAR-T cell monotherapy. The safety profile could be considered comparable to that described for CAR-T cell monotherapy.

PRIMO Monte Carlo software as a tool for commissioning of an external beam radiotherapy treatment planning system.

Background: The purpose was to validate the PRIMO Monte Carlo software to be used during the commissioning of a treatment planning system (TPS). Materials and methods: The Acuros XB v. 16.1 algorithm of the Eclipse was configured for 6 MV and 6 MV flattening-filter-free (FFF) photon beams, from a TrueBeam linac equipped with a high-definition 120-leaf multileaf collimator (MLC). PRIMO v. 0.3.64.1814 software was used with the phase space files provided by Varian and benchmarked against the reference dosimetry dataset published by the Imaging and Radiation Oncology Core-Houston (IROC-H). Thirty Eclipse clinical intensity-modulated radiation therapy (IMRT)/volumetric modulated arc therapy (VMAT) plans were verified in three ways: 1) using the PTW Octavius 4D (O4D) system; 2) the Varian Portal Dosimetry system and 3) the PRIMO software. Clinical validation of PRIMO was completed by comparing the simulated dose distributions on the O4D phantom against dose measurements for these 30 clinical plans. Agreement evaluations were performed using a 3% global/2 mm gamma index analysis. Results: PRIMO simulations agreed with the benchmark IROC-H data within 2.0% for both energies. Gamma passing rates (GPRs) from the 30 clinical plan verifications were (6 MV/6MV FFF): 99.4% ± 0.5%/99.9% ± 0.1%, 99.8% ± 0.4%/98.9% ± 1.4%, 99.7% ± 0.4%/99.7% ± 0.4%, for the 1), 2) and 3) verification methods, respectively. Agreement between PRIMO simulations on the O4D phantom and 3D dose measurements resulted in GPRs of 97.9% ± 2.4%/99.7% ± 0.4%. Conclusion: The PRIMO software is a valuable tool for dosimetric verification of clinical plans during the commissioning of the primary TPS.

Disease-associated mutations impacting BC-loop flexibility trigger long-range transthyretin tetramer destabilization and aggregation.

Hereditary transthyretin amyloidosis (ATTR) is an autosomal dominant disease characterized by the extracellular deposition of the transport protein transthyretin (TTR) as amyloid fibrils. Despite the progress achieved in recent years, understanding why different TTR residue substitutions lead to different clinical manifestations remains elusive. Here, we studied the molecular basis of disease-causing missense mutations affecting residues R34 and K35. R34G and K35T variants cause vitreous amyloidosis, whereas R34T and K35N mutations result in amyloid polyneuropathy and restrictive cardiomyopathy. All variants are more sensitive to pH-induced dissociation and amyloid formation than the wild-type (WT)-TTR counterpart, specifically in the variants deposited in the eyes amyloid formation occurs close to physiological pHs. Chemical denaturation experiments indicate that all the mutants are less stable than WT-TTR, with the vitreous amyloidosis variants, R34G and K35T, being highly destabilized. Sequence-induced stabilization of the dimer-dimer interface with T119M rendered tetramers containing R34G or K35T mutations resistant to pH-induced aggregation. Because R34 and K35 are among the residues more distant to the TTR interface, their impact in this region is therefore theorized to occur at long range. The crystal structures of double mutants, R34G/T119M and K35T/T119M, together with molecular dynamics simulations indicate that their strong destabilizing effect is initiated locally at the BC loop, increasing its flexibility in a mutation-dependent manner. Overall, the present findings help us to understand the sequence-dynamic-structural mechanistic details of TTR amyloid aggregation triggered by R34 and K35 variants and to link the degree of mutation-induced conformational flexibility to protein aggregation propensity.

Three simple steps to improve the interpretability of EEG-SVM studies.

Machine-learning systems that classify electroencephalography (EEG) data offer important perspectives for the diagnosis and prognosis of a wide variety of neurological and psychiatric conditions, but their clinical adoption remains low. We propose here that much of the difficulties translating EEG-machine-learning research to the clinic result from consistent inaccuracies in their technical reporting, which severely impair the interpretability of their often-high claims of performance. Taking example from a major class of machine-learning algorithms used in EEG research, the support-vector machine (SVM), we highlight three important aspects of model development (normalization, hyperparameter optimization, and cross-validation) and show that, while these three aspects can make or break the performance of the system, they are left entirely undocumented in a shockingly vast majority of the research literature. Providing a more systematic description of these aspects of model development constitute three simple steps to improve the interpretability of EEG-SVM research and, in fine, its clinical adoption.