Amplification-free CRISPR/Cas detection technology: challenges, strategies, and perspectives.

Rapid and accurate molecular diagnosis is a prerequisite for precision medicine, food safety, and environmental monitoring. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)-based detection, as a cutting-edged technique, has become an immensely effective tool for molecular diagnosis because of its outstanding advantages including attomolar level sensitivity, sequence-targeted single-base specificity, and rapid turnover time. However, the CRISPR/Cas-based detection methods typically require a pre-amplification step to elevate the concentration of the analyte, which may produce non-specific amplicons, prolong the detection time, and raise the risk of carryover contamination. Hence, various strategies for target amplification-free CRISPR/Cas-based detection have been developed, aiming to minimize the sensitivity loss due to lack of pre-amplification, enable detection for non-nucleic acid targets, and facilitate integration in portable devices. In this review, the current status and challenges of target amplification-free CRISPR/Cas-based detection are first summarized, followed by highlighting the four main strategies to promote the performance of target amplification-free CRISPR/Cas-based technology. Furthermore, we discuss future perspectives that will contribute to developing more efficient amplification-free CRISPR/Cas detection systems.

Sensitive, Single-Pot, Two-Stage Assay for Hepatitis Viruses.

When left untreated, hepatitis B virus (HBV) and hepatitis C virus (HCV) infections may cause severe illnesses. Since these infections remain asymptomatic for many years, routine screening of populations at risk is critical for therapy initiation. The current standard of care mandates a screening antibody test for HCV, followed by a confirmatory laboratory-based molecular test and treatment. Multiple visits to the clinic are inconvenient, and many patients fail to follow up. To address this challenge, we have developed sensitive, two-stage, isothermal molecular (Penn-RAMP) point-of-care tests to enable test and treat strategy. Penn-RAMP's first stage is comprised of recombinase polymerase amplification (RPA), while its second stage is comprised of loop-mediated isothermal amplification (LAMP). Penn-RAMP is more sensitive than LAMP or RPA alone. We designed a custom pre-LAMP buffer to maximize the volume of RPA products that can be added to the LAMP reaction mix without inhibition and forward and backward primers. Penn-RAMP was implemented in a single pot comprised of two compartments separated by a thermally removable barrier. RAMP's first stage is carried out above the barrier at the RPA incubation temperature. When the pot is heated to the LAMP incubation temperature, the barrier melts away, and the RPA reaction volume mixes with the pre-LAMP buffer, facilitating second-stage amplification. This entire process can be carried out with minimal instrumentation. Our HBV and HCV tests detect, respectively, as few as 10 and 25 virions within 30 min. The viral load can be estimated based on signal threshold time.

Manually-Operated, Slider Cassette for Multiplexed Molecular Detection at the Point of Care.

Effective control of epidemics, individualized medicine, and new drugs with virologic response-dependent dose and timing require, among other things, simple, inexpensive, multiplexed molecular detection platforms suitable for point of care and home use. Herein, we describe our progress towards developing such a platform that includes sample lysis, nucleic acid isolation, concentration, purification, and amplification. Our diagnostic device comprises a sliding component that houses the nucleic acid isolation membrane and a housing containing three amplification reaction chambers with dry stored reagents, blisters with buffers and wash solutions, and absorption pads to facilitate capillarity pull and waste storage. After sample introduction, the user slides the slider within the housing from one station to another to carry out various unit operations. The slider motion induces blisters to discharge their contents, effectuating washes, and eventual elution of captured nucleic acids into reaction chambers. The slider cassette mates with a processor that incubates isothermal amplification but can also be made to operate instrumentation-free. We demonstrate our cassette's utility for the co-detection of the human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV). These three blood-borne pathogens co-infect many people worldwide with severe personal and public health consequences.

Invited review: Sensor technologies for real-time monitoring of the rumen environment.

Quantifying digestive and fermentative processes within the rumen environment has been the subject of decades of research; however, our existing research methodologies preclude time-sensitive and spatially explicit investigation of this system. To better understand the temporal and spatial dynamics of the rumen environment, real-time and in situ monitoring of various chemical and physical parameters in the rumen through implantable microsensor technologies is a practical solution. Moreover, such sensors could contribute to the next generation of precision livestock farming, provided sufficient wireless data networking and computing systems are incorporated. In this review, various microsensor technologies applicable to real-time metabolic monitoring for ruminants are introduced, including the detection of parameters for rumen metabolism, such as pH, temperature, histamine concentrations, and volatile fatty acid concentrations. The working mechanisms and requirements of the sensors are summarized with respect to the selected target parameters. Lastly, future challenges and perspectives of this research field are discussed.

Long-Term Efficacy of T3 Versus T3+T4 Thoracoscopic Sympathectomy for Concurrent Palmar and Plantar Hyperhidrosis.

BACKGROUND: More than 50% of patients with palmar hyperhidrosis (PAH) also have plantar hyperhidrosis (PLH). We compared the long-term results of T3 sympathectomy with those of combined T3+T4 sympathectomy among patients with concurrent PAH and PLH. MATERIALS AND METHODS: We retrospectively analyzed the records of patients with concurrent PAH and PLH who underwent T3 alone or T3+T4 sympathectomy from January 1, 2012, to December 31, 2017. Preoperative and postoperative sweating (hyperhidrosis index) was evaluated through questionnaires, physical examination, and outpatient follow-up. The relief rates and hyperhidrosis index were used as outcome measures to compare the efficacy of the two approaches. Patients' satisfaction and side effects were also evaluated. RESULTS: Of the 220 eligible patients, 60 underwent T3 sympathectomy (T3 group), and 160 underwent T3+T4 sympathectomy (T3+T4 group). Compared with the T3 group, the T3+T4 group showed higher symptom relief rates both for PAH (98.75% versus 93.33%, P = 0.048) and PLH (65.63% versus 46.67%, P = 0.01), and a greater postoperative decrease in both hyperhidrosis indices. The rate of severe compensatory hyperhidrosis also increased (10% versus 5%, P = 0.197), although the rates of overall satisfaction were comparable between the groups. The incidence of postoperative pneumothorax requiring chest tube placement and postoperative neuralgia was also similar. There were no cases of perioperative death, secondary operation, wound infection, or Horner syndrome in either group. CONCLUSIONS: Compared with T3 alone, T3+T4 sympathectomy achieved a higher symptom relief rate and a lower hyperhidrosis index. T3+T4 sympathectomy may be a choice for the treatment of concurrent PAH and PLH; however, patients need to be informed that this kind of surgery may increase the risk of compensatory sweating.

Cascade Trisulfur Radical Anion (S3•-) Addition/Electron Detosylation Process for the Synthesis of 1,2,3-Thiadiazoles and Isothiazoles.

Trisulfur radical anion (S3•-) mediated reactions with in situ formed azoalkenes and α,ß-usaturated N-sulfonylimines for the construction of 1,2,3-thiadiazoles and isothiazoles has been developed. S3•- is in situ generated from potassium sulfide in DMF. These two approaches provide a new, safe, and simple way to construct 4-subsituted 1,2,3-thiadiazoles, 5-subsituted 1,2,3-thiadiazoles, and isothiazole in good yields. The reactions include the formation of the new C-S and N-S bonds via S3•- addition and electron detosylation under mild conditions.

Hydrothermal synthesis of single-crystalline perovskite PbTiO3 nanosheets with dominant (001) facets.

Single-crystalline tetragonal perovskite lead titanate (PbTiO3) nanosheets with dominant (001) facets have been successfully synthesized by employing layered K2Ti6O13 nanofibers as titanium sources. The as-prepared PbTiO3 nanosheets were characterized by means of X-ray diffraction, field-emission scanning electron microscopy, transmission electron microscopy (TEM), high-resolution TEM, and selected-area electron diffraction. In order to understand the formation mechanism of the PbTiO3 nanosheets, a series of time-dependent experiments were performed. Because of the substitution of Pb(2+) ions for K(+) ions, the TiO6 octahedral lamellas exfoliate from the layered K2Ti6O13 crystal structure. Then the exfoliated TiO6 octahedral lamellas as templates transform to lamellar PbTiO3 species by reacting with the dehydrated Pb(2+) ions. With hydrothermal treatment prolongation, the lamellar PbTiO3 species crystallize to single-crystalline PbTiO3 nanosheets. Moreover, the thickness of the synthesized single-crystalline PbTiO3 nanosheets can be tailored in the range of 10-50 nm by controlling the hydrothermal treatment time. In addition, the band gap and the optoelectronic properties of the single-crystalline PbTiO3 nanosheets are investigated by UV-vis absorption and photoluminescence.

Invited review: integration of technologies and systems for precision animal agriculture-a case study on precision dairy farming.

Precision livestock farming (PLF) offers a strategic solution to enhance the management capacity of large animal groups, while simultaneously improving profitability, efficiency, and minimizing environmental impacts associated with livestock production systems. Additionally, PLF contributes to optimizing the ability to manage and monitor animal welfare while providing solutions to global grand challenges posed by the growing demand for animal products and ensuring global food security. By enabling a return to the "per animal" approach by harnessing technological advancements, PLF enables cost-effective, individualized care for animals through enhanced monitoring and control capabilities within complex farming systems. Meeting the nutritional requirements of a global population exponentially approaching ten billion people will likely require the density of animal proteins for decades to come. The development and application of digital technologies are critical to facilitate the responsible and sustainable intensification of livestock production over the next several decades to maximize the potential benefits of PLF. Real-time continuous monitoring of each animal is expected to enable more precise and accurate tracking and management of health and well-being. Importantly, the digitalization of agriculture is expected to provide collateral benefits of ensuring auditability in value chains while assuaging concerns associated with labor shortages. Despite notable advances in PLF technology adoption, a number of critical concerns currently limit the viability of these state-of-the-art technologies. The potential benefits of PLF for livestock management systems which are enabled by autonomous continuous monitoring and environmental control can be rapidly enhanced through an Internet of Things approach to monitoring and (where appropriate) closed-loop management. In this paper, we analyze the multilayered network of sensors, actuators, communication, networking, and analytics currently used in PLF, focusing on dairy farming as an illustrative example. We explore the current state-of-the-art, identify key shortcomings, and propose potential solutions to bridge the gap between technology and animal agriculture. Additionally, we examine the potential implications of advancements in communication, robotics, and artificial intelligence on the health, security, and welfare of animals.

Precision technologies are revolutionizing animal agriculture by enhancing the management of animal welfare and productivity. To fully realize the potential benefits of precision livestock farming (PLF), the development and application of digital technologies are needed to facilitate the responsible and sustainable intensification of livestock production over the next several decades. Importantly, the digitalization of agriculture is expected to provide collateral benefits of ensuring audibility in value chains while assuaging concerns associated with labor shortages. In this paper, we analyze the multilayered network of sensors, actuators, communication, and analytics currently in use in PLF. We analyze the various aspects of sensing, communication, networking, and intelligence on the farm leveraging dairy farms as an example system. We also discuss the potential implications of advancements in communication, robotics, and artificial intelligence on the security and welfare of animals.

Molecular Detection of Infectious Laryngotracheitis Virus in Chickens with a Microfluidic Chip.

Infectious laryngotracheitis (ILT) is a viral disease of chickens' respiratory system that imposes considerable financial burdens on the chicken industry. Rapid, simple, and specific detection of this virus is crucial to enable proper control measures. Polymerase chain reaction (PCR)-based molecular tests require relatively expensive instruments and skilled personnel, confining their application to centralized laboratories. To enable chicken farms to take timely action and contain the spread of infection, we describe a rapid, simple, semi-quantitative benchtop isothermal amplification (LAMP) assay, and a field-deployable microfluidic device for the diagnosis of ILTV infection in chickens. Our assay performance was compared and favorably agreed with quantitative PCR (qPCR). The sensitivity of our real-time LAMP test is 250 genomic copies/reaction. Clinical performance of our microfluidic device using samples from diseased chickens showed 100% specificity and 100% sensitivity in comparison with benchtop LAMP assay and the gold-standard qPCR. Our method facilitates simple, specific, and rapid molecular ILTV detection in low-resource veterinary diagnostic laboratories and can be used for field molecular diagnosis of suspected ILT cases.

A portable, 3D printed, microfluidic device for multiplexed, real time, molecular detection of the porcine epidemic diarrhea virus, transmissible gastroenteritis virus, and porcine deltacoronavirus at the point of need.

The porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine deltacoronavirus (PDCoV) are emerging/reemerging coronaviruses (CoVs) of neonatal pigs that cause great economic losses to pig farms and pork processors. Specific, rapid, and simple multiplex detection of these viruses is critical to enable prompt implementation of appropriate control measures. Conventional methods for molecular diagnosis require skilled personnel and relatively sophisticated equipment, restricting their use in centralized laboratories. We developed a low-cost, rapid, semi-quantitative, field deployable, 3D-printed microfluidic device for auto-distribution of samples and self-sealing and real-time and reverse transcription-loop-mediated isothermal amplification (RT-LAMP), enabling the co-detection of PEDV, TGEV and PDCoV within 30 minutes. Our assay's analytical performance is comparable with a benchtop, real-time RT-LAMP assay and the gold standard quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay with limits of detection of 10 genomic copies per reaction for PEDV and PDCoV, and 100 genomic copies per reaction for TGEV. Evaluation of clinical specimens from diseased pigs with our microfluidic device revealed excellent concordance with both benchtop RT-LAMP and qRT-PCR. Our portable RT-LAMP microfluidic chip will potentially facilitate simple, specific, rapid multiplexed detection of harmful infections in minimally equipped veterinary diagnostic laboratories and on-site in pigs' farms.

Efficacy and safety of Chinese herbal compound in the treatment of functional constipation: A protocol for systematic review and meta-analysis.

BACKGROUND: Functional constipation refers to constipation without organic lesions caused by dietary factors, mood depression, changes in life rules, and poor bowel habits. Clinically, constipation is mainly manifested by changes of stool texture, difficulty or lack of bowel movement, and dry stool. Sometimes, it can be accompanied by abdominal distension and abdominal discomfort. Chinese herbal compound is a prescription which is composed of 2 or more medicinal flavors and is designed for relatively certain diseases and syndromes. Clinical studies have shown that TCM compounds have a good therapeutic effect on functional constipation, but there is no evidence of evidence-based medicine. The purpose of this study is to systematically evaluate the efficacy and safety of TCM compounds in the treatment of functional constipation, and to provide evidence-based basis for the clinical application of TCM compounds in the treatment of constipation. METHODS: A systematic search was performed on English database (PubMed, Embase, Web of Science, the Cochrane Library) and Chinese database (CNKI, Wanfang, Weipu (VIP), CBM), in addition to the manual retrieval of Baidu and Google academic for randomized controlled trials (RCTs) on the treatment of functional constipation with Chinese herbal compound. The retrieval time limit was from the establishment of the database to August 2020. Two researchers independently screened the literature, extracted the data and evaluated the quality of the included studies. A meta-analysis was performed using RevMan5.3 software. RESULTS: In this study, the efficacy and safety of TCM herbal compounds in the treatment of functional constipation were evaluated by the overall effective rate, recovery rate, colonic transmission function, and other indicators. CONCLUSIONS: This study will provide reliable evidence-based evidence for the clinical application of Chinese herbal compound in the treatment of functional constipation. OSF REGISTRATION NUMBER: DOI 10.17605/OSF.IO/D5ECF.

Membrane-sensitive bacterial DNA extractions and absolute quantitation of recovery efficiencies.

Absolute quantification of waterborne pathogens is mandatory for microbiological risk assessment (MRA). Determination of the DNA recovery efficiency is an essential step before the quantitative molecular measurements, which has been largely ignored. In this study, we compared the DNA recovery efficiency and quality of five extraction methods, including two modified phenol-chloroform-based extractions with mechanical shearing and three commercial kits for the extraction of DNA from indigenous mixed-bacteria culture of river water. All of the methods gave relatively satisfying results from the pelleted sample through centrifugation. However, the commercial kits provided surprisingly low DNA yields for membrane-filtered samples because of DNA trapping and/or absorption on the membrane. Integrating with enzymatic lysis, bath sonication, phenol extraction, and alcohol precipitation achieved highest DNA yields and an acceptable DNA integrity for quantitative PCR. A plasmid containing the human GADPH gene fragment was demonstrated to be a suitable spiking control for determining the absolute DNA recovery efficiency. The unexpectedly low efficiencies of commercial kit extractions imply the significant underestimation of pathogenic bacteria in previous studies, which should gain enough concern in the area of pathogen monitoring in the future.

Aerobic Oxidative Cycloaddition of α-Chlorotosylhydrazones with Arylamines: General Chemoselective Construction of 1,4-Disubstituted and 1,5-Disubstituted 1,2,3-Triazoles under Metal-Free and Azide-Free Conditions.

A novel synthetic approach toward 1,4-disubstituted 1,2,3-triazoles and 1,5-disubstituted 1,2,3-triazoles by aerobic oxidative cycloaddition of α-chlorotosylhydrazone with primary aryl amine has been developed. Significantly, the reaction proceeds smoothly to afford 1,4-disubstituted 1,2,3-triazoles and 1,5-disubstituted 1,2,3-triazoles under catalyst-free, metal-free, azide-free, and peroxide-free conditions.

Efficient multicomponent strategy to pentacyclic pyrazole-fused naphtho[1,8-fg]isoquinolines through cleavage of two carbon-carbon bonds.

Multicomponent reactions of acenaphthylene-1,2-dione with diaroylmethanes and electron-rich pyrazol-5-amines have been discovered. A series of new and polyfunctionalized pentacyclic pyrazole-fused naphtho[1,8-fg]isoquinolines were regioselectively synthesized. The reactions were easy to perform simply by mixing three common reactants in AcOH. During these reaction processes, the insertion of active methylene of diaroylmethane into the sp(2)-sp(2) C-C bond of the cyclohexa-2,5-dienone ring was readily achieved and two C-C bonds were cleaved under transition-metal-free conditions.