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Wastewater surveillance (WS), when coupled with advanced molecular techniques, offers near real-time monitoring of community-wide transmission of SARS-CoV-2 and allows assessing and mitigating COVID-19 outbreaks, by evaluating the total microbial assemblage in a community. Composite wastewater samples (24 h) were collected weekly from a manhole between December 2020 and November 2021 in Maryland, USA. RT-qPCR results showed concentrations of SARS-CoV-2 RNA recovered from wastewater samples reflected incidence of COVID-19 cases. When a drastic increase in COVID-19 was detected in February 2021, samples were selected for microbiome analysis (DNA metagenomics, RNA metatranscriptomics, and targeted SARS-CoV-2 sequencing). Targeted SARS-CoV-2 sequencing allowed for detection of important genetic mutations, such as spike: K417N, D614G, P681H, T716I, S982A, and D1118H, commonly associated with increased cell entry and reinfection. Microbiome analysis (DNA and RNA) provided important insight with respect to human health-related factors, including detection of pathogens and their virulence/antibiotic resistance genes. Specific microbial species comprising the wastewater microbiome correlated with incidence of SARS-CoV-2 RNA, suggesting potential association with SARS-CoV-2 infection. Climatic conditions, namely, temperature, were related to incidence of COVID-19 and detection of SARS-CoV-2 in wastewater, having been monitored as part of an environmental risk score assessment carried out in this study. In summary, the wastewater microbiome provides useful public health information, and hence, a valuable tool to proactively detect and characterize pathogenic agents circulating in a community. In effect, metagenomics of wastewater can serve as an early warning system for communicable diseases, by providing a larger source of information for health departments and public officials.

Celiac disease (CD) comprises over 1% of the world’s population and is a chronic multisystem immune-mediated condition manifested by digestive and/or extradigestive symptoms caused by food intake of gluten. This review looked at the risk of children diagnosed with CD developing SARS-CoV-2 infection and possible severe forms of COVID-19. A better understanding of the interaction and effects of SARS-CoV-2 infection in CD is very important, as is the role of environmental and genetic factors, but especially the molecular mechanisms involved in modulating intestinal permeability with impact on autoimmunity. CD inspired the testing of a zonulin antagonist for the fulminant form of multisystem inflammatory syndrome in children (MIS-C) and paved the way for the discovery of new molecules to regulate the small intestine barrier function and immune responses. Original published works on COVID-19 and CD, new data and points of view have been analyzed because this dangerous virus SARS-CoV-2 is still here and yet influencing our lives. Medical science continues to focus on all uncertainties triggered by SARS-CoV-2 infection and its consequences, including in CD. Although the COVID-19 pandemic seems to be gradually extinguishing, there is a wealth of information and knowledge gained over the last two years and important life lessons to analyze, as well as relevant conclusions to be drawn to deal with future pandemics. Zonulin is being studied extensively in immunoengineering as an adjuvant to improving the absorption of new drugs and oral vaccines.

Mechanical anisotropy is an essential property for biomolecules to assume structural and functional roles in mechanobiology. However, there is insufficient information on the mechanical anisotropy of ligand-biomokcule complexes. Herein, we investigated the mechanical property of individual human telomeric G-quadruplexes bound with a telomestatin ligand by optical tweezers. Stacking of the ligand to the G-tetradplanes changes the conformation of the G-quadruplex, which resembles a balloon squeezed at certain directions. Such a squeezed balloon effect strengthens the G-tetrad planes whereas dislocates and weakens the loops in the G-quadruplex upon ligand binding. These dynamic interactions indicate that the binding between the ligand and G-quadruplex follows the induced-fit model. We anticipate that the altered mechanical anisotropy of the ligand-G-quadruplex complex can add additional level of regulations on the motor enzymes that process DNA or RNA molecules.

Rubber is a fascinating material in both industry and daily life. The development of elastomeric material in nanotechnology is imperative due to its economic and technological potential. By virtue of their distinctive physicochemical properties, nucleic acids have been extensively explored in material science. The Phi29 DNA packaging motor contains a 3WJ with three angles of 97°, 125°, and 138°. Here, the rubber-like property of RNA architectures was investigated using optical tweezers and in vivo imaging technologies. The 3WJ 97° interior angle was contracted or stretched to 60°, 90°, and 108° at will to build elegant RNA triangles, squares, pentagons, cubes, tetrahedrons, dendrimers, and prisms. RNA nanoarchitecture was stretchable and shrinkable by optical tweezer with multiple extension and relaxation repeats like a rubber. Comparing to gold and iron nanoparticles with the same size, RNA nanoparticles display stronger cancer-targeting outcomes, while less accumulation in healthy organs. Generally, the upper limit of renal excretion is 5.5 nm; however, the 5, 10, and 20 nm RNA nanoparticles passed the renal filtration and resumed their original structure identified in urine. These findings solve two previous mysteries: (1) Why RNA nanoparticles have an unusually high tumor targeting efficiency since their rubber or amoeba-like deformation property enables them to squeeze out of the leaky vasculature to improve the EPR effect; and (2) why RNA nanoparticles remain non-toxic since they can be rapidly cleared from the body via renal excretion into urine with little accumulation in the body. Considering its controllable shape and size plus its rubber-like property, RNA holds great promises for industrial and biomedical applications especially in cancer therapeutics delivery.

Objective To develop a real-time monitoring system that has the potential to guide the titration of anesthetic agents in the treatment of pediatric status epilepticus (SE). Methods We analyzed stored multichannel electroencephalographic (EEG) data collected from 12 pediatric patients with generalized SE. EEG recordings were initially segmented in 500 ms time-windows. Features characterizing the power, frequency, and entropy of the signal were extracted from each segment. The segments were annotated as bursts (B), suppressions (S), or artifacts (A) by two electroencephalographers. The EEG features together with the annotations were inputted in a three-layer feed forward neural network (NN). The sensitivity and specificity of NNs with different architectures and training algorithms to classify segments into B, S, or A were estimated. Results The maximum sensitivity (95.96% for B, 89.25% for S, and 75% for A) and specificity (89.36 for B, 96.26% for S, and 99.8% for A) was observed for the NN with 10 nodes in the hidden layer. By using this NN, we designed a real-time system that estimates the burst-suppression index (BSI). Conclusions Our system provides a reliable real-time estimate of multichannel BSI requiring minimal memory and computation time. Significance The system has the potential to assist intensive care unit attendants in the continuous EEG monitoring.

G-Quadruplex and i-motif are tetraplex structures that may form in opposite strands at the same location of a duplex DNA. Recent discoveries have indicated that the two tetraplex structures can have conflicting biological activities, which poses a challenge for cells to coordinate. Here, by performing innovative population analysis on mechanical unfolding profiles of tetraplex structures in double-stranded DNA, we found that formations of G-quadruplex and i-motif in the two complementary strands are mutually exclusive in a variety of DNA templates, which include human telomere and promoter fragments of hINS and hTERT genes. To explain this behavior, we placed G-quadruplex- and i-motif-hosting sequences in an offset fashion in the two complementary telomeric DNA strands. We found simultaneous formation of the G-quadruplex and i-motif in opposite strands, suggesting that mutual exclusivity between the two tetraplexes is controlled by steric hindrance. This conclusion was corroborated in the BCL-2 promoter sequence, in which simultaneous formation of two tetraplexes was observed due to possible offset arrangements between G-quadruplex and i-motif in opposite strands. The mutual exclusivity revealed here sets a molecular basis for cells to efficiently coordinate opposite biological activities of G-quadruplex and i-motif at the same dsDNA location.

GOALS The goal of this study was to evaluate the impact of inpatient outcomes of gastrointestinal bleeding (GIB) related to percutaneous coronary intervention (PCI). BACKGROUND With all-cause mortality increasing in patients undergoing PCIs, outcomes for GIB associated with PCI may be adversely impacted. STUDY Using the National Inpatient Sample (2007 to 2012), we performed a nested case-control study assessing inpatient outcomes including incidence and mortality for PCI-related GIB hospitalizations. Multivariate logistic regression analyses were performed to determine significant predictors for GIB incidence and mortality. RESULTS A total of 9332 (1.2%) of PCI hospitalizations were complicated by GIB with the age-adjusted incidence rate increasing 13% from 2007 (11.3 GIB per 1000 PCI) to 2012 (12.8). Patients ≥75 years of age experienced the steepest incline in GIB incidence, which increased 31% during the study period. Compared with non-GIB patients, mean length of stay (9.4 d vs. 3.3 d) and median cost of care ($29,236 vs. $17,913) was significantly higher. Significant demographic risk factors for GIB included older age and comorbid risk factors included gastritis or duodenitis, and Helicobacter pylori infection.In total, 1044 (11%) of GIB patients died during hospitalization with the GIB mortality rate increasing 30% from 2007 (95 deaths per 1000 GIB) to 2012 (123). Older age had the strongest association with inpatient mortality. CONCLUSIONS Inpatient incidence and mortality for PCI-related GIB has been increasing particularly with a large increase in incidence among older patients. A multidisciplinary approach focused on risk-stratifying patients may improve preventable causes of GIB.

The 3' human telomeric overhang provides ample opportunities for the formation and interaction of G-quadruplexes, which have shown impacts on many biological functions including telomerase activities in the telomere region. However, in the few investigations on DNA constructs that approach to the full length of the human telomeric overhang, the presence of higher-order quadruplex-quadruplex interactions is still a subject of debate. Herein, we employed dynamic splint ligation (DSL) to prepare a DNA construct, 5'-(TTAGGG)24 or 24G, which has the length comparable to the full stretch of 3' human telomeric overhang. Using mechanical unfolding assays in laser tweezers, we observed a minor population (∼5%) of higher-order interactions between G-quadruplexes, while the majority of the quadruplexes follow the bead-on-a-string model. Analyses on the noninteracting G-quadruplexes in the 24G construct showed features similar to those of the stand-alone G-quadruplexes in the 5'-(TTAGGG)4 (4G) construct. As each 24G construct contains as many as six G-quadruplexes, this method offers increased throughput for the time-consuming mechanical unfolding experiments of non-B DNA structures.

Miscued communication often leads to misfolding and aggregation of the proteins involved in many diseases. Owing to the ensemble average property of conventional techniques, detailed communication diagrams are difficult to obtain. Mechanical unfolding affords an unprecedented perspective on cooperative transitions by observing a protein along a trajectory defined by two mutated cysteine residues. Nevertheless, this approach requires tedious sample preparation at the risk of altering native protein conformations. To address these issues, we applied click chemistry to tether a protein to the two dsDNA handles through primary amines in lysine residues as well as at the N terminus. As a proof of concept, we used laser tweezers to mechanically unfold and refold calmodulin along 36 trajectories, maximally allowed by this strategy in a single batch of protein preparation. Without a priori knowledge of the particular residues to which the double-stranded DNA handles attach, we used hierarchical cluster analysis to identify 20 major trajectories, according to the size and the pattern of unfolding transitions. We dissected the cooperativity into all-or-none and partially cooperative events, which represent strong and weak high-order interactions in proteins, respectively. Although the overall cooperativity is higher within the N or C lobe than that between the lobes, the all-or-none cooperativity is anisotropic among different the unfolding trajectories and becomes relatively more predominant when the size of the protein segments increases. The average cooperativity for all-or-none transitions falls within the expected range observed by ensemble techniques, which supports the hypothesis that unfolding of a free protein can be reconstituted from individual trajectories.

In investigating the binding interactions between the human telomeric RNA (TERRA) G-quadruplex (GQ) and its ligands, it was found that the small molecule carboxypyridostatin (cPDS) and the GQ-selective antibody BG4 simultaneously bind the TERRA GQ. We previously showed that the overall binding affinity of BG4 for RNA GQs is not significantly affected in the presence of cPDS. However, single-molecule mechanical unfolding experiments revealed a population (48 %) with substantially increased mechanical and thermodynamic stability. Force-jump kinetic investigations suggested competitive binding of cPDS and BG4 to the TERRA GQ. Following this, the two bound ligands slowly rearrange, thereby leading to the minor population with increased stability. Given the relevance of G-quadruplexes in the regulation of biological processes, we anticipate that the unprecedented conformational rearrangement observed in the TERRA-GQ-ligand complex may inspire new strategies for the selective stabilization of G-quadruplexes in cells.

We developed a 375-channel, whole-head magnetoencephalography (MEG) system (“BabyMEG”) for studying the electrophysiological development of human brain during the first years of life. The helmet accommodates heads up to 95% of 36-month old boys in the USA. The unique two-layer sensor array consists of: (1) 270 magnetometers (10 mm diameter, ∼15 mm coil-to-coil spacing) in the inner layer, (2) thirty-five three-axis magnetometers (20 mm × 20 mm) in the outer layer 4 cm away from the inner layer. Additionally, there are three three-axis reference magnetometers. With the help of a remotely operated position adjustment mechanism, the sensor array can be positioned to provide a uniform short spacing (mean 8.5 mm) between the sensor array and room temperature surface of the dewar. The sensors are connected to superconducting quantum interference devices (SQUIDs) operating at 4.2 K with median sensitivity levels of 7.5 fT/√Hz for the inner and 4 fT/√Hz for the outer layer sensors. SQUID outputs are digitized by a 24-bit acquisition system. A closed-cycle helium recycler provides maintenance-free continuous operation, eliminating the need for helium, with no interruption needed during MEG measurements. BabyMEG with the recycler has been fully operational from March, 2015. Ongoing spontaneous brain activity can be monitored in real time without interference from external magnetic noise sources including the recycler, using a combination of a lightly shielded two-layer magnetically shielded room, an external active shielding, a signal-space projection method, and a synthetic gradiometer approach. Evoked responses in the cortex can be clearly detected without averaging. These new design features and capabilities represent several advances in MEG, increasing the utility of this technique in basic neuroscience as well as in clinical research and patient studies.

Recent experiments provided controversial observations that either parallel or non-parallel G-quadruplex exists in molecularly crowded buffers that mimic cellular environment. Here, we used laser tweezers to mechanically unfold structures in a human telomeric DNA fragment, 5′-(TTAGGG)4TTA, along three different trajectories. After the end-to-end distance of each unfolding geometry was measured, it was compared with PDB structures to identify the best-matching G-quadruplex conformation. This method is well-suited to identify biomolecular structures in complex settings not amenable to conventional approaches, such as in a solution with mixed species or at physiologically significant concentrations. With this approach, we found that parallel G-quadruplex coexists with non-parallel species (1:1 ratio) in crowded buffers with dehydrating cosolutes [40% w/v dimethyl sulfoxide (DMSO) or acetonitrile (ACN)]. In crowded solutions with steric cosolutes [40% w/v bovine serum albumin (BSA)], the parallel G-quadruplex constitutes only 10% of the population. This difference unequivocally supports the notion that dehydration promotes the formation of parallel G-quadruplexes. Compared with DNA hairpins that have decreased unfolding forces in crowded (9 pN) versus diluted (15 pN) buffers, those of G-quadruplexes remain the same (20 pN). Such a result implies that in a cellular environment, DNA G-quadruplexes, instead of hairpins, can stop DNA/RNA polymerases with stall forces often

Disturbances of the autonomic nervous system (ANS) are common in neuropsychiatric disorders. Disease specific alterations of both sympathetic and parasympathetic activity can be assessed by heart rate variability (HRV), whereas electrodermal activity (EDA) can assess sympathetic activity. In posttraumatic stress disorder (PTSD), parasympathetic HRV parameters are typically decreased and EDA is increased, whereas in major depressive disorder (MDD) and dissociation, both parasympathetic and sympathetic markers are decreased. ANS abnormalities have also been identified in psychogenic nonepileptic seizures (PNES) by using HRV, indicating lower parasympathetic activity at baseline. In addition to reviewing the current literature on ANS abnormalities in PTSD, MDD, and disorders with prominent dissociation, including borderline personality disorder (BPD), this article also presents data from a pilot study on EDA in patients with PNES. Eleven patients with PNES, during an admission to our epilepsy monitoring unit (EMU), were compared with 9 with generalized tonic-clonic seizures (GTCS). The area under the EDA curve, the number of EDA responses lasting longer than 2 seconds, and the number of EDA surges during sleep (sympathetic sleep storms) were calculated on ictal and interictal days by an automated algorithm. EDA changes in PNES patients did not follow a systematic pattern of sympathetic hyperarousal (like EDA after GTCS) but were more variable. How specific PNES semiologies, and/or underlying neuropsychiatric disorders, may influence ictal and interictal EDA patterns, and lead to a novel diagnostic biomarker remains to be evaluated in future larger studies.

The repair of atrioventricular valves requires optimal valve exposure through even unconventional surgical approaches. This is especially true in the situation of multiple re-do valve surgeries, where extensive adhesions may prevent adequate valve exposure. When multiple valve surgeries are contemplated, an extensive incisional approach is required without causing damage to any surrounding crucial structures. Re-do surgeries are described in patients with prosthetic valve endocarditis where the correction of all valves involved is necessary, as is the correction of underlying ventricular pathologies such as sinuses and atrioventricular tunnels. As several surgical approaches have been attempted in these situations, a technique is described which involves a superior biatrial approach after transecting the aorta, which addresses the mitral, tricuspid and aortic valves and allows the correction of associated ventricular pathologies.

A patient who had a small bowel mesentery perforation following insertion of a suprapubic catheter (SPC) is described. He had no bowel complaints immediately following the procedure, but presented 10 weeks later with insidious onset bowel obstruction due to the kink caused by the catheter. This complication occurred despite cystoscopy control and adequate bladder distension prior to the procedure. This isolated case illustrates the fact that regardless of the ease and frequency of SPC insertion, complications do occur.

Single-stranded guanine (G)-rich sequences at the 3' end of human telomeres provide ample opportunities for physiologically relevant structures, such as G-quadruplexes, to form and interconvert. Population equilibrium in this long sequence is expected to be intricate and beyond the resolution of ensemble-average techniques, such as circular dichroism, NMR, or X-ray crystallography. By combining a force-jump method at the single-molecular level and a statistical population deconvolution at the sub-nanometer resolution, we reveal a complex population network with unprecedented transition dynamics in human telomeric sequences that contain four to eight TTAGGG repeats. Our kinetic data firmly establish that G-triplexes are intermediates to G-quadruplexes while long-loop G-quadruplexes are misfolded population minorities whose formation and disassembly are faster than G-triplexes or regular G-quadruplexes. The existence of misfolded DNA supports the emerging view that structural and kinetic complexities of DNA can rival those of RNA or proteins. While G-quadruplexes are the most prevalent species in all the sequences studied, the abundance of a misfolded G-quadruplex in a particular telomeric sequence decreases with an increase in the loop length or the number of long-loops in the structure. These population patterns support the prediction that in the full-length 3' overhang of human telomeres, G-quadruplexes with shortest TTA loops would be the most dominant species, which justifies the modeling role of regular G-quadruplexes in the investigation of telomeric structures.

Polytetrafluoroethylene or Gore-Tex sutures are used for chordal replacement, and are durable but extremely slippery, a property which causes knot slippage and ineffective neochordal length. We report a modification that is simple and reproducible in preventing knot slippage and maintaining optimal neochordal length, without the use of devices or additional sutures.

Congenital aneurysm of the body of the left atrium causing severe mitral incompetence is extremely rare. The cause of mitral incompetence has been attributed to a distortion of the mitral annulus by the aneurysm, or to coexisting pathology in the mitral valve apparatus. Surgical techniques to treat this condition include excision of the aneurysm along with mitral valve repair or replacement. Herein is described the case of a 30-year-old man with large aneurysm of the left atrial body and severe mitral regurgitation (MR) treated by endoatrial patch plasty. The mitral incompetence was due to prolapse of the posterior mitral annulus into the neck of the aneurysm. Under cardiopulmonary bypass, the neck of the aneurysm was closed using a Dacron patch. Endoaneurysmorrhaphy with a Dacron patch effectively restored the mitral competence and simultaneously excluded the aneurysm from the left atrium. To further strengthen the posterior mitral annulus, a mitral annuloplasty was added using a rigid Carpentier-Edwards ring. The patient was anticoagulated with warfarin for six weeks after surgery. Transthoracic echocardiography performed at a six-month follow up confirmed the absence of any residual aneurysm or MR. To the authors' knowledge, this is the first case of left atrial aneurysm with severe MR to be treated in this way.

Letters not directly related to articles published in Clinical Medicine and presenting unpublished original data should be submitted for publication in this section. Clinical and scientific letters should not exceed 500 words and may include one table and up to five references. The central theme of