Multiplexed In Vivo Screening Using Barcoded Aptamer Technology to Identify Oligonucleotide-Based Targeting Reagents.

Recent FDA approvals of mRNA vaccines, short-interfering RNAs, and antisense oligonucleotides highlight the success of oligonucleotides as therapeutics. Aptamers are excellent affinity reagents that can selectively label protein biomarkers, but their clinical application has lagged. When formulating a given aptamer for in vivo use, molecular design details can determine biostability and biodistribution; therefore, extensive postselection manipulation is often required for each new design to identify clinically useful reagents harboring improved pharmacokinetic properties. Few methods are available to comprehensively screen such aptamers, especially in vivo, constituting a significant bottleneck in the field. In this study, we introduce barcoded aptamer technology (BApT) for multiplexed screening of predefined aptamer formulations in vitro and in vivo. We demonstrate this technology by simultaneously investigating 20 aptamer formulations, each harboring different molecular designs, for targeting Non-Small Cell Lung Cancer cells and tumors. Screening in vitro identified a 45 kDa bispecific formulation as the best cancer cell targeting reagent, whereas screening in vivo identified a 30 kDa monomeric formulation as the best tumor-specific targeting reagent. The multiplexed analysis pipeline also identified biodistribution phenotypes shared among formulations with similar molecular architectures. The BApT approach we describe here has the potential for broad application to fields where oligonucleotide-based targeting reagents are desired.

Vesicocutaneous fistula at site of closed suprapubic tube tract as the first presenting sign of giant bladder stone.

Bladder stones represent approximately 5% of all cases of urolithiasis and are typically identified and managed long before causing irreversible renal injury. We present a case of a man in his 40s with a prior history of a gunshot wound to the abdomen who presented with leakage from a previously healed suprapubic tube tract and was found to have a giant bladder stone with a resulting renal injury. He subsequently underwent a combined open cystolithotomy and vesicocutaneous fistulotomy during his hospitalisation, which helped to improve his renal function. In addition to there being few reported cases of bladder stones >10 cm, this represents the first report in the literature of an associated decompressive 'pop-off' mechanism through a fistulised tract.

Analysis of microscopy techniques to measure segregation in continuous-cast steel slabs.

The accurate characterisation of centreline segregation requires precise measurements of composition variations over large length scales (10 - 1 $^{-1}$ m ${\rm {m}}$ ) across the centreline of the cast product, while having high resolution, sufficient to quantify the significant composition variations between dendrites due to microsegregation at very small length scales (10 - 5 m $^{-5}{\rm {m}}$ ). This study investigates the potential of a novel microscopy technique, named Synchrotron Micro X-ray Flurorescence (SMXRF), to generate large-scale high-resolution segregation maps from a steel sample taken from a thin slab caster. Two methods, Point Analysis and Regression Analysis, are proposed for SMXRF data calibration. By comparing with the traditional Laser-Induced Breakdown Spectroscopy (LIBS), and Electron Probe Micro Analyser (EPMA) techniques, we show that SMXRF is successful in quantitative characterisation of centreline segregation. Over large areas (e.g. 12 × $\times$ 16 mm 2 ${\rm {mm}}^2$ ) and at high resolution (10-50 µ m $\mu\text{m}$ pixel size) various techniques yield comparable outcomes in terms of composition maps and solute profiles. The findings also highlight the importance of both high spatial resolution and large field of view to have a quantitative, accurate, and efficient measurement tool to investigate segregation phenomena.

Renal mass imaging modalities: does body mass index (BMI) matter?

PURPOSE: Accurate measurement of renal mass size is crucial in the management of renal cancer. With the burdensome cost of imaging yet its need for management, a better understanding of the variability among patients when determining mass size remains of urgent importance. Current guidelines on optimal imaging are limited, especially with respect to body mass index (BMI). The aim of this study is to discern which modalities accurately measure renal mass size and whether BMI influences such accuracy. METHODS: A multi-institutional chart review was performed for adult patients undergoing partial or radical nephrectomy between 2018 and 2021, with 236 patients ultimately included. Patients were categorized by BMI (BMI 1: 18.5-24.9, BMI 2: 25-29.9, BMI 3: 30-34.9, and BMI 4: ≥ 35). The greatest mass lengths were compared between the pathology report and the following: computerized tomography (CT), renal ultrasound, and magnetic resonance imaging (MRI). RESULTS: The difference between greatest length on CT with contrast and MRI were significantly different when compared to pathologic measurement. BMI groups 3 and 4 were found to have a significant difference in size estimates compared to BMI 2 for CT with contrast. No difference was found between size estimates by BMI group for any other imaging modality. CONCLUSION: CT with contrast becomes less accurate at estimating mass size for patients with BMI > 30. While contrast-enhanced CT remains a vital imaging modality for tissue enhancement in the context of unknown renal masses, caution must be used for mass size estimation in the obese population.

Vesicoureteral reflux severity is not associated with unplanned urinary catheterization or length of hospital stay after ureteroneocystostomy.

INTRODUCTION: Vesicoureteral reflux (VUR) is a common urologic condition affecting approximately 1% of all children. Surgical success often depends on the grade of VUR, as patients with grades 4 or 5 have been have a greater risk for postoperative complications. Unplanned urinary catheter placement (UCP) postoperatively and prolonged length of hospital stay (LOS) are indicative of unexpected complications. The association between VUR severity and such metrics remain unclear. OBJECTIVE: The study's objective is to determine if the severity of VUR is associated with higher rates of UCP or prolonged LOS after ureteroneocystostomy (UNC). STUDY DESIGN: The 2020 National Surgical Quality Improvement Program Pediatric database was analyzed for patients with VUR. A total of 1742 patients were initially evaluated with 1373 meeting exclusion criteria. The patients were divided into 3 groups of varying voiding cystourethrogram (VCUG) or radionuclide cystogram (RNC) severity: VCUG Grade 1 or RNC Grade 1 (Group A), VCUG Grade 2 or 3 or RNC Grade 2 (Group B), and VCUG Grade 4 or 5 or RNC Grade 3 (Group C). Basic statistical analysis was performed, and logistic regression was performed with both UCP and LOS as dependent variables. RESULTS: Among the 1373 patients, 2.9% were included in Group A, 32.5% were in Group B, and 64.6% were in Group C. Significant differences were found among the groups for mean age, gender, inpatient status, rate of congenital malformation, ureteral stents, and ASA classification. Regarding surgical treatment, differences were also found comparing mean operative time, LOS, laterality and type of procedure, urine culture results, rates of UTI, surgical site infections, postoperative returns to the emergency department, and unplanned procedures and catheterization. Multivariate analysis demonstrated no significant association between the rate of UCP and VUR severity, while postoperative UTI and unplanned procedure were both independent factors associated with UCP postoperatively. Additionally, postoperative UTI, ASA classification, mean operation time, ureteral stent placement, unplanned procedure, and UCP were independent factors found to contribute to LOS. CONCLUSION: Greater VUR severity does not appear to increase the need for catheterization or prolong hospital stay, while the development of a UTI postoperatively or having an additional unplanned procedure are associated with an increased likelihood of both. The postoperative course after UNC also appears to be influenced more so by other factors such as the operative approach and whether complications arise.

Preoperative and Intraoperative Urine Cultures and Its Association With Postoperative Infection After Ureteroneocystostomy.

OBJECTIVE: To unveil this association, we hypothesize that preoperative and intraoperative urinary tract infection (UTI) will be correlated with postoperative UTI and sepsis occurrence. PATIENTS AND METHODS: The 2020 National Surgical Quality Improvement Program Pediatric (NSQIP-P) data was analyzed for patients undergoing ureteroneocystostomy (UNC) for vesicoureteral reflux (VUR). Patients that underwent UNC for treatment of VUR with urine culture obtained within 2weeks preoperatively or on the day of surgery were identified. The patients were divided into 3 groups: no bacterial growth, bacterial growth with UTI, and bacterial growth polymicrobial growth. Patient demographics and preoperative variables were evaluated. RESULTS: The postoperative urinary tract infection rate of the three groups were 2.0%, 9.2%, and 9.9% for group A, B, C, respectively (P < .001). Postoperative sepsis was noted to be 0.5%, 1.3%, and 3.6% for group A, B, C (P < .01). Additionally, there was a difference between mean operative time (P < .001), mean length of stay (P = .03), and mean days from operation to discharge (P < .01). On adjusted analysis, both groups B and C had higher rates of UTI compared to group A. Group C was also seen to have greater rates of sepsis on adjusted analysis. CONCLUSION: The association found between preoperative UTI with less than 2 species of microorganisms (group B) and postoperative UTI indicates that UTI treatment and antibiotic prophylaxis should be considered when undergoing UNC for VUR. The results of this study may lead to more careful consideration of the use of preoperative and intraoperative urine culture as well as treatment of UTI in pediatric patients with VUR undergoing UNC.

CasPEDIA Database: a functional classification system for class 2 CRISPR-Cas enzymes.

CRISPR-Cas enzymes enable RNA-guided bacterial immunity and are widely used for biotechnological applications including genome editing. In particular, the Class 2 CRISPR-associated enzymes (Cas9, Cas12 and Cas13 families), have been deployed for numerous research, clinical and agricultural applications. However, the immense genetic and biochemical diversity of these proteins in the public domain poses a barrier for researchers seeking to leverage their activities. We present CasPEDIA (http://caspedia.org), the Cas Protein Effector Database of Information and Assessment, a curated encyclopedia that integrates enzymatic classification for hundreds of different Cas enzymes across 27 phylogenetic groups spanning the Cas9, Cas12 and Cas13 families, as well as evolutionarily related IscB and TnpB proteins. All enzymes in CasPEDIA were annotated with a standard workflow based on their primary nuclease activity, target requirements and guide-RNA design constraints. Our functional classification scheme, CasID, is described alongside current phylogenetic classification, allowing users to search related orthologs by enzymatic function and sequence similarity. CasPEDIA is a comprehensive data portal that summarizes and contextualizes enzymatic properties of widely used Cas enzymes, equipping users with valuable resources to foster biotechnological development. CasPEDIA complements phylogenetic Cas nomenclature and enables researchers to leverage the multi-faceted nucleic-acid targeting rules of diverse Class 2 Cas enzymes.

Targeting lung cancer with clinically relevant EGFR mutations using anti-EGFR RNA aptamer.

A significant fraction of non-small cell lung cancer (NSCLC) cases are due to oncogenic mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR). Anti-EGFR antibodies have shown limited clinical benefit for NSCLC, whereas tyrosine kinase inhibitors (TKIs) are effective, but resistance ultimately occurs. The current landscape suggests that alternative ligands that target wild-type and mutant EGFRs are desirable for targeted therapy or drug delivery development. Here we evaluate NSCLC targeting using an anti-EGFR aptamer (MinE07). We demonstrate that interaction sites of MinE07 overlap with clinically relevant antibodies targeting extracellular domain III and that MinE07 retains binding to EGFR harboring the most common oncogenic and resistance mutations. When MinE07 was linked to an anti-c-Met aptamer, the EGFR/c-Met bispecific aptamer (bsApt) showed superior labeling of NSCLC cells in vitro relative to monospecific aptamers. However, dual targeting in vivo did not improve the recognition of NSCLC xenografts compared to MinE07. Interestingly, biodistribution of Cy7-labeled bsApt differed significantly from Alexa Fluor 750-labeled bsApt. Overall, our findings demonstrate that aptamer formulations containing MinE07 can target ectopic lung cancer without additional stabilization or PEGylation and highlights the potential of MinE07 as a targeting reagent for the recognition of NSCLC harboring clinically relevant EGFRs.

Is Urethral Catheterization Necessary in Retzius-sparing Robot-assisted Radical Prostatectomy?

OBJECTIVE: To analyze whether urethral catheter (UC)-free anastomosis during Retzius-sparing radical prostatectomy (RP) results in worsening immediate perioperative and postoperative complications. METHODS: We retrospectively reviewed records of patients undergoing robotic-assisted RP with or without indwelling UC placement by a single surgeon between January 2020 and March 2022. Clinical and pathological characteristics were evaluated. An independent t-test was used to compare continuous variables, and Pearson's chi-square test or Fisher exact test was used to compare categorical variables. RESULTS: One hundred fifteen patients underwent robotic prostatectomy from January 2020 to June 2022. Sixty-two patients had both a UC for 12 hours postoperatively and a suprapubic catheter for 6days, while in 53 patients a UC was omitted. There was no significant difference noted between the groups regarding age, body mass index, American Society of Anesthesiology score, prostate-specific antigen, stage, node status, or positive margins. There were no significant differences in the rates of anastomotic leak, ileus, or urethral stricture. Patients had a significantly higher pad-free rate in the suprapubic catheter-only group (P = .04) at 3months. There was no difference in average number of pads used or the number using more than 1 pad/day. CONCLUSION: Omission of urethral catheterization during Retzius-sparing RP is safe and doesn't result in an increased risk in perioperative or postoperative complications, but rather appears to be associated with a significantly improved 3-month pad-free rate.

Relación de las Enfermedades Crónicas no Transmisibles y Ejercicio Físico en la Población Chilena entre los años 2015-2016 / Risk of Retention of Secretions in Patients with Invasive Mechanical Ventilation

Introducción. Las prevalencias de la inactividad física, el sedentarismo y el sobrepeso y obesidad han aumentado sus índices durante los últimos años en Chile, lo que conlleva al desarrollo y aparición de diversas enfermedades crónicas no transmisibles, como por ejemplo, hipertensión arterial, diabetes mellitus e inclusive enfermedades respiratorias, las cuales repercuten negativamente en la población y deterioran la calidad de vida de las personas, independientemente el sexo y el rango etario. El ejercicio físico es una de las principales herramientas utilizadas por diversos profesionales de la salud como método de prevención y tratamiento en la población afectada, inclusive representa una alternativa de menor costo. Objetivo. Relacionar el ejercicio físico de alta y mediana intensidad con las enfermedades crónicas no transmisibles en personas mayores a 18 años residentes en Chile durante el periodo 2015-2016. Métodos. Análisis cuantitativo, descriptivo, transversal y retrospectivo de base secundaria ENCAVI 2015-2016, de residentes en Chile mayores de 18 años y con al menos una de las siguientes condiciones de salud: Hipertensión arterial, Diabetes Mellitus y enfermedades respiratorias crónicas no transmisibles. Resultados. Todas las variables presentan relaciones significativas (p<0,005) (HTA, DM, enfermedades respiratorias, edad, horas sentado, ejercicio físico de moderada y alta intensidad. Sin embargo, las variables de ejercicio físico moderada intensidad y enfermedades respiratorias (p=0,578) y las variables de sexo y horas sentado (p=0,005) no presentan relación significativa. Discusión. El ejercicio físico de moderada intensidad es el que tiene mejor respuesta ante las diferentes patologías según diferentes autores, por otro lado, en la recopilación de datos podemos encontrar que este tipo de ejercicio es efectivo en la Diabetes Mellitus. Conclusiones. Según los datos analizados, mientras mayor frecuencia de ejercicio físico de alta intensidad (en días) y ejercicio físico de moderada intensidad (en horas) se realice durante los últimos días de la semana, los diagnósticos de las enfermedades crónicas no transmisibles (HTA, DM) y enfermedades respiratorias se verán controladas, mientras que, cuando existe menor frecuencia del ejercicio físico realizado durante los últimos días de la semana, tiende a existir mayor diagnóstico.

Background. The prevalence of physical inactivity, sedentary lifestyle, and overweight and obesity have increased their rates in recent years in Chile, which leads to the development and appearance of various chronic non-communicable diseases, such as for example, arterial hypertension, diabetes mellitus and even respiratory diseases, which have a negative impact on the population and deteriorate the quality of life of people, regardless of gender and age range (7). Physical exercise is one of the main tools used by various health professionals as a method of prevention and treatment in the affected population, even representing a lower cost alternative. Objetive. To relate high and medium intensity physical exercise with chronic non-communicable diseases in people over 18 years of age residing in Chile during the period 2015-2016. Methods. Quantitative, descriptive, cross-sectional and retrospective analysis of the ENCAVI 2015-2016 secondary base of residents in Chile over 18 years of age and with at least one of the following health conditions: High blood pressure, Diabetes Mellitus and chronic non-communicable respiratory diseases. Results. All the variables present significant relationships (p<0.005) (HTN, DM, respiratory diseases, age, hours sitting, moderate and high intensity physical exercise. However, the variables of moderate intensity physical exercise and respiratory diseases (p=0.578) and the variables of sex and sitting hours (p=0.005) do not present a significant relationship. Discussion. Moderate intensity physical exercise is the one that has the best response to different pathologies according to different authors, on the other hand, in data collection we can found that this type of exercise is effective in Diabetes Mellitus. Conclusions. According to the data analyzed, the greater the frequency of high-intensity physical exercise (in days) and moderate-intensity physical exercise (in hours) performed during the last days of the week, the diagnoses of chronic non-communicable diseases (HTN, DM) and respiratory diseases will be controlled, while, when there is less frequency of physical exercise carried out during the last days of the week, there tends to be a greater diagnosis.

Preservation of whole antibodies within ancient teeth.

Archaeological remains can preserve some proteins into deep time, offering remarkable opportunities for probing past events in human history. Recovering functional proteins from skeletal tissues could uncover a molecular memory related to the life-history of the associated remains. We demonstrate affinity purification of whole antibody molecules from medieval human teeth, dating to the 13th-15th centuries, from skeletons with different putative pathologies. Purified antibodies are intact retaining disulphide-linkages, are amenable to primary sequences analysis, and demonstrate apparent immunoreactivity against contemporary EBV antigen on western blot. Our observations highlight the potential of ancient antibodies to provide insights into the long-term association between host immune factors and ancient microbes, and more broadly retain a molecular memory related to the natural history of human health and immunity.

Novel and Engineered Type II CRISPR Systems from Uncultivated Microbes with Broad Genome Editing Capability.

Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 nucleases have been extensively used in biotechnology and therapeutics. However, many applications are not possible owing to the size, targetability, and potential off-target effects associated with currently known systems. In this study, we identified thousands of CRISPR type II effectors by mining an extensive, genome-resolved metagenomics database encompassing hundreds of thousands of microbial genomes. We developed a high-throughput pipeline that enabled us to predict tracrRNA sequences, to design single guide RNAs, and to demonstrate nuclease activity in vitro for 41 newly described subgroups. Active systems represent an extensive diversity of protein sequences and guide RNA structures and require diverse protospacer adjacent motifs (PAMs) that collectively expand the known targeting capability of current systems. Several nucleases showed activity levels comparable to or significantly higher than SpCas9, despite being smaller in size. In addition, top systems exhibited low levels of off-target editing in mammalian cells, and PAM-interacting domain engineered chimeras further expanded their targetability. These newly discovered nucleases are attractive enzymes for translation into many applications, including therapeutics.

Novel CRISPR-Associated Gene-Editing Systems Discovered in Metagenomic Samples Enable Efficient and Specific Genome Engineering.

Development of medicines using gene editing has been hampered by enzymological and immunological impediments. We described previously the discovery and characterization of improved, novel gene-editing systems from metagenomic data. In this study, we substantially advance this work with three such gene-editing systems, demonstrating their utility for cell therapy development. All three systems are capable of reproducible, high-frequency gene editing in primary immune cells. In human T cells, disruption of the T cell receptor (TCR) alpha-chain was induced in >95% of cells, both paralogs of the TCR beta-chain in >90% of cells, and >90% knockout of ß2-microglobulin, TIGIT, FAS, and PDCD1. Simultaneous double knockout of TRAC and TRBC was obtained at a frequency equal to that of the single edits. Gene editing with our systems had minimal effect on T cell viability. Furthermore, we integrate a chimeric antigen receptor (CAR) construct into TRAC (up to ∼60% of T cells), and demonstrate CAR expression and cytotoxicity. We next applied our novel gene-editing tools to natural killer (NK) cells, B cells, hematopoietic stem cells, and induced pluripotent stem cells, generating similarly efficient cell-engineering outcomes including the creation of active CAR-NK cells. Interrogation of our gene-editing systems' specificity reveals a profile comparable with or better than Cas9. Finally, our nucleases lack preexisting humoral and T cell-based immunity, consistent with their sourcing from nonhuman pathogens. In all, we show these new gene-editing systems have the activity, specificity, and translatability necessary for use in cell therapy development.

Global assessment of the sensitivity of water storage to hydroclimatic variations.

Observing basin water storage response due to hydroclimatic fluxes and human water use provides valuable insight to the sensitivity of water storage to climate change. Quantifying basin water storage changes due to climate and human water use is critical for water management yet remains a challenge globally. Observations from the Gravity Recovery and Climate Experiment (GRACE) mission are used to extract monthly available water (AW), representing the combined storage changes from groundwater and surface water stores. AW is combined with hydroclimatic fluxes, including precipitation (P) and evapotranspiration (ET) to quantify the hydroclimatic elasticity of AW for global basins. Our results detect consequential global water sensitivity to changes in hydroclimatic fluxes, where 25 % of land areas exhibit hydroclimatic elasticity of AW >10, implying that a 1 % change in monthly P-ET would result in a 10 % change in AW. Corroboration using a Budyko-derived metric substantiates our findings, demonstrating that basin water storage resilience to short-term water deficits is linked to basin partitioning predictability, and uniform seasonality of hydroclimatic fluxes. Our study demonstrates how small shifts in hydroclimate flux may affect available water storage potentially impacting billions globally.

Role of the endocannabinoid system in fragile X syndrome: potential mechanisms for benefit from cannabidiol treatment.

Multiple lines of evidence suggest a central role for the endocannabinoid system (ECS) in the neuronal development and cognitive function and in the pathogenesis of fragile X syndrome (FXS). This review describes the ECS, its role in the central nervous system, how it is dysregulated in FXS, and the potential role of cannabidiol as a treatment for FXS. FXS is caused by deficiency or absence of the fragile X messenger ribonucleoprotein 1 (FMR1) protein, FMRP, typically due to the presence of >200 cytosine, guanine, guanine sequence repeats leading to methylation of the FMR1 gene promoter. The absence of FMRP, following FMR1 gene-silencing, disrupts ECS signaling, which has been implicated in FXS pathogenesis. The ECS facilitates synaptic homeostasis and plasticity through the cannabinoid receptor 1, CB1, on presynaptic terminals, resulting in feedback inhibition of neuronal signaling. ECS-mediated feedback inhibition and synaptic plasticity are thought to be disrupted in FXS, leading to overstimulation, desensitization, and internalization of presynaptic CB1 receptors. Cannabidiol may help restore synaptic homeostasis by acting as a negative allosteric modulator of CB1, thereby attenuating the receptor overstimulation, desensitization, and internalization. Moreover, cannabidiol affects DNA methylation, serotonin 5HT1A signal transduction, gamma-aminobutyric acid receptor signaling, and dopamine D2 and D3 receptor signaling, which may contribute to beneficial effects in patients with FXS. Consistent with these proposed mechanisms of action of cannabidiol in FXS, in the CONNECT-FX trial the transdermal cannabidiol gel, ZYN002, was associated with improvements in measures of social avoidance, irritability, and social interaction, particularly in patients who are most affected, showing ≥90% methylation of the FMR1 gene.

Attenuated Total Reflection Fourier Transform Infrared (ATR FT-IR) Spectroscopy Sensitivity to the Thermal Decay of Bone Collagen.

The analysis of collagen stability is of interest in forensics, archaeology, and molecular paleontology. Collagen decay rates are often measured by thermal kinetic studies that employ liquid chromatography mass spectrometry (LC-MS) to assay collagen quantities. However, these kinetic studies generally focus on measuring the decreasing levels of collagen instead of an exact molecular concentration of each sample. Thus, attenuated total reflection Fourier transform infrared (ATR FT-IR) spectroscopy can offer a simpler and less expensive alternative to LC-MS. The application of a new protocol to determine decreasing amounts of bone collagen in artificially decayed porcine and bovine bone was assessed. The protocol uses a forensic application of ATR FT-IR spectroscopy on size-restricted bone powder from three uniformly high temperature conditions. Also, for the first time, collagen-specific second-harmonic generation (SHG) imaging was also applied to artificially aged bone to add an independent, qualitative perspective to parallel FT-IR assessments. SHG images and ATR FT-IR spectra together reveal the same orderly bone collagen decay as found in previous thermal kinetic studies. Resulting Arrhenius plots with r2 values > 0.95 suggest that the ATR FT-IR-based protocol has potential as a precise and simple tool for measuring bone collagen decay rates. The results are significant for applications of thermal kinetic studies, and our protocol can serve as an inexpensive, precise, and pragmatic means of evaluating bone collagen stability within an array of conditions.

Alternate Graft Options for Staged Flexor Tendon Reconstruction: A Cadaveric Study of Hamstring Autografts Compared to Conventional Autografts.

PURPOSE: To compare the semitendinosus and gracilis tendon lengths and diameters to the palmaris longus, plantaris, flexor digitorum profundus, and flexor pollicis longus (FPL) tendons in a cadaveric model to evaluate the feasibility of hamstring autograft use for staged flexor tendon reconstruction. METHODS: Fifteen fresh cadavers were evaluated for surgical incisions about the knee, forearm, and hand. All flexor digitorum profundus (FDP), FPL, palmaris longus, plantaris, semitendinosus, and gracilis tendons were harvested from each specimen. Diameter and length were recorded and means with SDs were calculated. The mean diameters of the gracilis and semitendinosus were compared to the mean diameters of the FDP and FPL tendons. The hamstring tendon lengths were then compared in terms of percentage of the palmaris longus and plantaris tendon lengths. RESULTS: The gracilis (18.0 cm) and semitendinosus (19.9 cm) means were notably longer than the palmaris longus (16.0 cm) and shorter than the plantaris (30.0 cm). The average gracilis tendon diameter (3.8 mm) was smaller than the flexor tendon diameters except for the little finger FDP (3.8 mm). The semitendinosus tendon diameter (4.8 mm) was larger than all flexor tendons with the exception of the middle finger FDP (4.6 mm). Average gracilis and semitendinosus tendon diameters were 3.7 mm and 4.5 mm in males, and 3.8 mm and 4.8 mm in females. CONCLUSIONS: This study showed the gracilis tendon to have adequate length and diameter for potential autograft use in staged flexor tendon reconstruction in all digits but the little finger. The semitendinosus is larger in diameter than the native flexor tendons, making it a poor autograft option in cases with an intact pulley system. CLINICAL RELEVANCE: Common tendon autograft options for flexor tendon reconstruction are variably present, and the use of gracilis and semitendinosus autograft present potential graft options.

Aggregation and Oligomerization Characterization of ß-Lactoglobulin Protein Using a Solid-State Nanopore Sensor.

Protein aggregation is linked to many chronic and devastating neurodegenerative human diseases and is strongly associated with aging. This work demonstrates that protein aggregation and oligomerization can be evaluated by a solid-state nanopore method at the single molecule level. A silicon nitride nanopore sensor was used to characterize both the amyloidogenic and native-state oligomerization of a model protein ß-lactoglobulin variant A (ßLGa). The findings from the nanopore measurements are validated against atomic force microscopy (AFM) and dynamic light scattering (DLS) data, comparing ßLGa aggregation from the same samples at various stages. By calibrating with linear and circular dsDNA, this study estimates the amyloid fibrils' length and diameter, the quantity of the ßLGa aggregates, and their distribution. The nanopore results align with the DLS and AFM data and offer additional insight at the level of individual protein molecular assemblies. As a further demonstration of the nanopore technique, ßLGa self-association and aggregation at pH 4.6 as a function of temperature were measured at high (2 M KCl) and low (0.1 M KCl) ionic strength. This research highlights the advantages and limitations of using solid-state nanopore methods for analyzing protein aggregation.

Compact Cas9d and HEARO enzymes for genome editing discovered from uncultivated microbes.

Programmable, RNA-guided nucleases are diverse enzymes that have been repurposed for biotechnological applications. However, to further expand the therapeutic application of these tools there is a need for targetable systems that are small enough to be delivered efficiently. Here, we mined an extensive genome-resolved metagenomics database and identified families of uncharacterized RNA-guided, compact nucleases (between 450 and 1,050 aa). We report that Cas9d, a new CRISPR type II subtype, contains Zinc-finger motifs and high arginine content, features that we also found in nucleases related to HEARO effectors. These enzymes exhibit diverse biochemical characteristics and are broadly targetable. We show that natural Cas9d enzymes are capable of genome editing in mammalian cells with >90% efficiency, and further engineered nickase variants into the smallest base editors active in E. coli and human cells. Their small size, broad targeting potential, and translatability suggest that Cas9d and HEARO systems will enable a variety of genome editing applications.