bluuebunny/crossref_metadata_2025 huggingface dataset:
Magnitude and associated factors of intraoperative hypothermia among pediatric patients undergoing elective surgery at Ayder Comprehensive Specialized Hospital 2023: prospective observational cross-sectional study Tigray, Ethiopia
Authors: Bitsa Yihdego , Belay Aregawi , Mulaw Abera , Hadush Yared , Teferi Molla
Published: May 2024

Background:

Hypothermia is characterized by a drop in core body temperature of less than 36°C. It occurs frequently throughout the operating period and affects surgical patient outcomes differently in terms of morbidity and mortality. Because of coagulopathy, metabolic acidosis, multiple organ failure, hemodynamic instability, and infections, a core temperature below 34°C is strongly associated with mortality.

Objective:

This study aimed to assess the magnitude and associated factors of intraoperative hypothermia in pediatric patients undergoing elective surgery at the Ayder Comprehensive Specialized Hospital.

Method:

A prospective observational cross-sectional study was conducted on 399 pediatric patients undergoing elective surgery at Ayder Comprehensive Specialized Hospital in Tigray, Ethiopia, from 1 May 2023, to 30 July 2023. Participants in the study were selected by a systematic random sampling technique. The data collection procedure was chart review and intraoperative temperature measurement, and the collected data were analyzed by SPSS version 23. The independent variables were analyzed using binary and multi-logistic regression. The odds ratio, 95% CI, and p value of less than 0.05 were considered statistical significance.

Result:

The magnitude of intraoperative hypothermia was 52.9%. Neonate and infant [adjusted odds ratio (AOR): 6, 95% CI: 3.7, 9.8], (AOR=4.5, 95% CI: 2.9, 7) respectively, volume of fluid administered greater than half-liter [AOR: 4.37, (95% CI, 3, 6.4)], patients who underwent surgery during the morning [AOR: 5.3, (95% CI: 3.8, 7.4)], and duration of surgery and anesthesia greater than 120 minutes [AOR: 2.7, (95% CI, 1.8, 4)] and (AOR=3.4, 95% CI, 2.4, 4.9], respectively, were factors significantly associated with intraoperative hypothermia.

Conclusion and recommendation:

This study revealed a high magnitude of intraoperative hypothermia among pediatric patients. Being neonates and infants, having a cold volume of IV fluid administered greater than half a liter, entering surgery during the morning, the duration of surgery, and the anesthesia time were significantly associated with intraoperative hypothermia. The authors would like to advise anesthetists to use warm intravenous fluids, calculate IV fluids, and maintain room temperature.
NSAIDs for early management of acute respiratory infections
Authors: Bassetti Matteo , Andreoni Massimo , Santus Pierachille , Scaglione Francesco
Published: May 2024

Purpose of review

To review the rationale for and the potential clinical benefits of an early approach to viral acute respiratory infections with NSAIDs to switch off the inflammatory cascade before the inflammatory process becomes complicated.

Recent findings

It has been shown that in COVID-19 as in other viral respiratory infections proinflammatory cytokines are produced, which are responsible of respiratory and systemic symptoms. There have been concerns that NSAIDs could increase susceptibility to SARS-CoV-2 infection or aggravate COVID-19. However, recent articles reviewing experimental research, observational clinical studies, randomized clinical trials, and meta-analyses conclude that there is no basis to limit the use of NSAIDs, which may instead represent effective self-care measures to control symptoms.

The inflammatory response plays a pivotal role in the early phase of acute respiratory tract infections (ARTIs); a correct diagnosis of the cause and a prompt therapeutic approach with NSAIDs may have the potential to control the pathophysiological mechanisms that can complicate the condition, while reducing symptoms to the benefit of the patient. A timely treatment with NSAIDs may limit the inappropriate use of other categories of drugs, such as antibiotics, which are useless when viral cause is confirmed and whose inappropriate use is responsible for the development of resistance.
Papillary Thyroid Cancer, Small Cell Lung Cancer, and Parathyroid Adenoma Synchronously Visualized on 99mTc-MIBI SPECT/CT in a Patient With Hyperparathyroidism
Authors: Chun In Kook , Lee Seungkoo
Published: May 2024

99mTc-MIBI scintigraphy is a nuclear medicine imaging modality commonly used for the preoperative localization of parathyroid adenomas in patients with hyperparathyroidism. In addition, 99mTc-MIBI can also be used for imaging various tumors due to its unique mechanism of intracellular accumulation. Here, we introduced a case of a single 99mTc-MIBI SPECT/CT simultaneously visualized two different malignant tumors, such as papillary thyroid cancer and small cell lung cancer, along with a parathyroid adenoma in a patient with hyperparathyroidism. The clinical usefulness of 99mTc-MIBI SPECT/CT was also explored by comparing it with 18F-FDG PET/CT among the three tumors.
Adult Neuroblastoma of the Neck Is Better Imaged With 18F-FDG PET/CT Than With 18F-DOPA PET/CT
Authors: Alabed Yazan Z.
Published: May 2024

A 41-year-old woman presented with 2 months history of right submandibular swelling. Biopsy revealed neuroblastoma (NB). Patient was referred for staging PET/CT scan. We compared the findings of 18F-DOPA PET/CT and 18F-FDG PET/CT scans. Both imaging modalities were positive in the patient; however, tumor delineation was superior with 18F-FDG PET/CT. Tumor uptake of FDG was significantly higher than tumor uptake of DOPA. Follow-up FDG PET/CT scan postsurgery showed local recurrent NB and their metastases avidly concentrate FDG. We present a very rare case of adult NB of the neck better imaged with FDG instead of DOPA PET/CT.
Secondary Procedures on the Palate to Correct/Improve Speech: Past, Present and Future
Authors: Cohen Mimis
Published: May 2024

Speech production in general and in patients with cleft palate in particular is multifactorial. In addition to the complex velopharyngeal mechanism, all structures of the oral cavity have some contribution for correct speech production. Therefore, in addition to the velopharynx the surgeons and other cleft/craniofacial team members need to perform a thorough and complete evaluation of all structures to fully appreciate the causative factor(s) responsible for inadequate speech production after palatoplasty and to prescribe the most appropriate, personalized management plan. The purpose of this communication is to stress the importance of areas other than the velopharyngx that could have a negative impact on speech. More specifically, the issues of palatoplasty failures and palatal revisions will be presented. This is an area of significant importance and could represent the first line of defense before considering procedures altering the anatomy of the velopharynx, such as pharyngeal flaps, pharyngoplasties, and others. Issues covering the effects of skeletal and dental problems, such as malocclusion, partial or complete edentulism can also affect speech but are outside the scope of this communication.
Indications, Stability, and Surgical Outcomes of Using Osteochondral Nasal Septal Graft as a Septal Extension Graft in Rhinoplasty Patients: A Prospective Cohort Study
Authors: Assiri Hassan , Alarfaj Ahmed , Aloraini Nouf , AlNassar Raad
Published: May 2024

Septal extension graft (SEG) is an adaptable tool that controls, supports, and stabilizes the nasal tip projection, rotation, length, and shape. Recent studies comparing SEGs to more conventional columellar strut grafts found that SEGs provided improved results. This prospective cohort study included 66 patients divided into 2 groups based on their indications. Group 1 was patients who underwent septorhinoplasty using osteochondral septal extension graft (OSEG), and group 2 underwent septorhinoplasty using cartilaginous SEG. The main reason for undergoing OSEG was insufficient septal cartilage (P<0.001). The outcomes were not significantly different between both groups, with the % of patients who experienced positive outcomes ranging from 78.8% to 97%. The OSEG is a safe, valid, and effective option in septorhinoplasty that can be used in patients with insufficient cartilaginous septum.
Does Time to Asystole in Donors After Circulatory Death Impact Recipient Outcome in Liver Transplantation?
Authors: Malik Abdullah K. , Tingle Samuel J. , Varghese Chris , Owen Ruth , Mahendran Balaji , Figueiredo Rodrigo , Amer Aimen O. , Currie Ian S. , White Steven A. , Manas Derek M. , Wilson Colin H.
Published: May 2024

Background.

The agonal phase can vary following treatment withdrawal in donor after circulatory death (DCD). There is little evidence to support when procurement teams should stand down in relation to donor time to death (TTD). We assessed what impact TTD had on outcomes following DCD liver transplantation.

Methods.

Data were extracted from the UK Transplant Registry on DCD liver transplant recipients from 2006 to 2021. TTD was the time from withdrawal of life-sustaining treatment to asystole, and functional warm ischemia time was the time from donor systolic blood pressure and/or oxygen saturation falling below 50 mm Hg and 70%, respectively, to aortic perfusion. The primary endpoint was 1-y graft survival. Potential predictors were fitted into Cox proportional hazards models. Adjusted restricted cubic spline models were generated to further delineate the relationship between TTD and outcome.

Results.

One thousand five hundred fifty-eight recipients of a DCD liver graft were included. Median TTD in the entire cohort was 13 min (interquartile range, 9–17 min). Restricted cubic splines revealed that the risk of graft loss was significantly greater when TTD ≤14 min. After 14 min, there was no impact on graft loss. Prolonged hepatectomy time was significantly associated with graft loss (hazard ratio, 1.87; 95% confidence interval, 1.23-2.83; P = 0.003); however, functional warm ischemia time had no impact (hazard ratio, 1.00; 95% confidence interval, 0.44-2.27; P > 0.9).

Conclusions.

A very short TTD was associated with increased risk of graft loss, possibly because of such donors being more unstable and/or experiencing brain stem death as well as circulatory death. Expanding the stand down times may increase the utilization of donor livers without significantly impairing graft outcome.
Molecular mechanisms promoting long-term cytopenia after BCMA CAR-T therapy in Multiple Myeloma
Authors: Palacios-Berraquero Maria Luisa , Rodriguez-Marquez Paula , Calleja-Cervantes Maria Erendira , Berastegi Nerea , Zabaleta Aintzane , Burgos Leire , Alignani Diego , San Martin-Uriz Patxi , Vilas-Zornoza Amaia , Rodriguez-Diaz Saray , Inoges Susana , Lopez-Diaz de Cerio Ascensión , Huerga Sofia , Tamariz Esteban , Rifón Jose , Alfonso-Pierola Ana , Lasarte Juan Jose , Paiva Bruno , Hernaez Mikel , Rodriguez-Otero Paula , San Miguel Jesus , Ezponda Teresa , Rodriguez-Madoz Juan Roberto , Prosper Felipe
Published: May 2024

Hematological toxicity is a common side effect of CAR-T therapies, being particularly severe in relapsed/refractory multiple myeloma (MM) patients. In this study, we analyzed a cohort of 48 patients treated with BCMA CAR-T cells to characterize the kinetics of cytopenia, identify predictive factors and determine potential mechanism underlying these toxicities. The overall incidence of cytopenia was 95.74%, and grade>3 thrombocytopenia and neutropenia one month after infusion was observed in 57% and 53% of the patients and was still present after 1 year in 4 and 3 patients respectively. Presence of cytopenia at baseline and high peak inflammatory markers highly correlated with cytopenia persisting up to three months. To determine potential mechanisms underpinning cytopenias, we evaluated the paracrine effect of BCMA CAR-T cells on the differentiation of HSPCs using anex-vivomyeloid differentiation model. Phenotypic analysis showed that supernatants from activated CAR-T cells (spCAR) halted HSPCs differentiation promoting more immature phenotypes, with reduced expression of granulocytic, monocytic and erythroid markers. Single-cell RNAseq demonstrated an upregulation of transcription factors associated with early stages of hematopoietic differentiation in the presence of spCAR (GATA2, RUNX1andCEBPA)and decreased activity of key regulons involved in neutrophil and monocytic maturation (ID2andMAFB). Our results suggest that CAR-T cell activation negatively influences hematopoietic differentiation through paracrine effects inducing arrest of HSPCs maturation and contributes to the understanding of severe cytopenia observed after CAR-T cell treatment in MM patients. These results may identify regulatory mechanisms involved in alter hematopoiesis and could lead to alternative therapeutic strategies.

KEY POINTS

Long-lasting cytopenia after BCMA CAR-T therapy correlates with baseline cytopenia and peak inflammatory markers.

Supernatants from activated BCMA CAR-T cells induced an inhibition of ex-vivo myeloid differentiation and rewiring of transcriptional programs associated with hematopoietic differentiation.
An anti-SARS-CoV-2 formula consisting volatile oil from TCM through inhibiting multiple targets in cultured cells
Authors: Zhang Huan , Guo Wuyan , Shen Jing , Song Xiaorui , Zhang Bo
Published: May 2024

At present, coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variant virus are prevalent all over the world, taking a major toll on human lives worldwide. Oral mucosa and saliva are the high-risk routes of transmission. Inactivation of the virus in the mouth is one of the important strategies to reduce the source infectivity of the virus. However, the current preparations for inactivating oral virus are mainly aimed at ACE2 protein, and its effect needs to be improved. In this study, through literature mining, multi-target screening and other methods, we screened and optimized the volatile oil formula (Artemisiae argyi folium, Chrysanthemum morifolium, Trollii chinensis flos, Lonicerae japonicae flos) from the volatile oil of traditional Chinese medicine to inhibit the invasion and replication of SARS-CoV-2. A test using pseudotype virus with S glycoprotein confirmed that this formula could effectively bind to the S glycoprotein to prevent SARS-CoV-2 host cell entry. Molecular docking experiments showed that the m ultiple molecules in the formula might weakly bind to these targets including ACE2, cathepsin L, furin, mpro/3cl. In all, the volatile oil formula designed in this paper offers a general affordable strategy to protect patients from SARS-CoV-2 infections through debulking or minimizing transmission to others.
Development of fluoro-7-aminocarboxycoumarin-based mitochondrial pyruvate carrier inhibitors as anticancer agents
Authors: Schumacher Tanner J. , Gardner Zachary S. , Rumbley Jon , Ronayne Conor T. , Mereddy Venkatram R.
Published: May 2024

Reprogrammed metabolism of cancer cells offers a unique target for pharmacological intervention. In the current study, a series of novel and potentially metabolically stable fluoro-substituted aminocarboxycoumarin derivatives are evaluated for their mitochondrial pyruvate carrier (MPC) inhibition properties. Our studies indicate that the aminocarboxycoumarin template elicits potent MPC inhibitory characteristics, and specifically, structure activity relationship studies show that theN-methyl-N-benzyl structural template provides the optimal inhibitory capacity. Further respiratory experiments demonstrate that candidate compounds specifically inhibit pyruvate driven respiration without substantially affecting other metabolic fuels consistent with MPC inhibition. Further, computational homology and inhibitor docking studies illustrate that aminocarboxycoumarin binding characteristics are indicative of reversible covalent bonding with amino acids in the pyruvate binding domain. Epifluorescent microscopy experiments illustrated that FACC2 accumulates in the mitochondria to a similar extent as parent 7ACC2. Additionally, lead candidate aminocarboxycoumarin derivative D7 elicits cancer cell proliferation inhibition specifically in monocarboxylate transporter 1 (MCT1) expressing 4T1, consistent with its ability to accumulate intracellular lactate.In vivotumor growth studies illustrate that D7 significantly reduces the tumor burden in two isogeneic murine cell lines 4T1 and 67nr. These studies provide novel MPC inhibitors with potential for anticancer applications.
Predicting Future Kinetic States of Physicochemical Systems Using Generative Pre-trained Transformer
Authors: Bera Palash , Mondal Jagannath
Published: May 2024

Capturing the time evolution and predicting future kinetic states of physicochemical systems present significant challenges due to the precision and computational effort required. In this study, we demonstrate that the transformer, a machine learning model renowned for machine translation and natural language processing, can be effectively adapted to predict the dynamical state-to-state transition kinetics of biologically relevant physicochemical systems. Specifically, by using sequences of time-discretized states from Molecular Dynamics (MD) simulation trajectories as input, we show that a transformer can learn the complex syntactic and semantic relationships within the trajectory. This enables this generative pre-trained transformer (GPT) to predict kinetically accurate sequences of future states for a diverse set of models and biomolecules of varying complexity. Remarkably, the GPT can predict future states much faster than traditional MD simulations. We show that it is particularly adept at forecasting the time evolution of an out-of-equilibrium active system that do not maintain detailed balance. An analysis of self-attention mechanism inherent in transformers is found to hold crucial role for capturing the long-range correlations necessary for accurate state-to-state transition predictions. Together, our results highlight the ability of transformer based machine learning model in generating future states of physicochemical systems with statistical precision.
Structure, Biology, and Applications of Filamentous Bacteriophages
Authors: Rakonjac Jasna , Gold Vicki A.M. , León-Quezada Rayén I. , Davenport Catherine H.
Published: May 2024

The closely relatedEscherichia coliFf filamentous phages (f1, fd, and M13) have taken a fantastic journey over the past 60 years, from the urban sewerage from which they were first isolated, to their use in high-end technologies in multiple fields. Their relatively small genome size, high titers, and the virions that tolerate fusion proteins make the Ffs an ideal system for phage display. Folding of the fusions in the oxidizing environment of theE. coliperiplasm makes the Ff phages a platform that allows display of eukaryotic surface and secreted proteins, including antibodies. Resistance of the Ffs to a broad range of pH and detergents facilitates affinity screening in phage display, whereas the stability of the virions at ambient temperature makes them suitable for applications in material science and nanotechnology. Among filamentous phages, only the Ffs have been used in phage display technology, because of the most advanced state of knowledge about their biology and the various tools developed forE. colias a cloning host for them. Filamentous phages have been thought to be a rather small group, infecting mostly Gram-negative bacteria. A recent discovery of more than 10 thousand diverse filamentous phages in bacteria and archaea, however, opens a fascinating prospect for novel applications. The main aim of this review is to give detailed biological and structural information to researchers embarking on phage display projects. The secondary aim is to discuss the yet-unresolved puzzles, as well as recent developments in filamentous phage biology, from a viewpoint of their impact on current and future applications.
The pComb3 Phagemid Family of Phage Display Vectors
Authors: Rader Christoph
Published: May 2024

A phagemid is a plasmid that contains the origin of replication and packaging signal of a filamentous phage. Following bacterial transformation, a phagemid can be replicated and amplified as a plasmid, using a double-stranded DNA origin of replication, or it can be replicated as single-stranded DNA for packaging into filamentous phage particles. The use of phagemids enables phage display of large proteins, such as antibody fragments. Phagemid pComb3 was among the first phage display vectors used for the generation and selection of antibody libraries in the 50-kDa Fab format, a monovalent proxy of natural antibodies. Affording a robust and versatile tool for more than three decades, phage display vectors of the pComb3 phagemid family have been widely used for the discovery, affinity maturation, and humanization of antibodies in Fab, scFv, and single-domain formats from naive, immune, and synthetic antibody repertoires. In addition, they have been used for broadening phage display to the mining of nonimmunoglobulin repertoires. This review examines conceptual, functional, and molecular features of the first-generation phage display vector pComb3 and its successors, pComb3H, pComb3X, and pC3C.
Ex Vivo Brain Imaging inDrosophila
Authors: Boto Tamara , Tomchik Seth M.
Published: May 2024

Analysis of neuronal circuit function inDrosophilacan be facilitated with an ex vivo imaging preparation. In this approach, the brain is isolated but intact, preserving neuronal connectivity and function. The preparation has several advantages, including stability, accessibility for pharmacological manipulations, and the ability to image over several hours. The full range of genetic approaches available inDrosophilacan be readily combined with pharmacological manipulations in this preparation, and numerous genetically encoded reporters are available to image cellular events, ranging from Ca2+signaling to neurotransmitter release.
Filleting and Immunostaining of Larvae to VisualizeDrosophilaDendritic Arborization Neuron Dendrite Arbors
Authors: Xu Fangke , Tann Jason Y. , Wilkes Oliver R. , Moore Adrian W.
Published: May 2024

Nervous system formation involves the specification of neuron identity, followed by precise circuit construction; this includes controlling the pattern and connectivity of the dendrite arbor.Drosophiladendritic arborization (da) neurons are a powerful experimental model for studying dendrite arbor differentiation mechanisms. da neuron dendrite arbors elaborate in two dimensions in the body wall, making it easy to visualize them with high resolution. Immunostaining is a conventional method to examine arbor pattern and the subcellular distribution of proteins. In addition, images acquired from immunostaining protocols can amplify weaker signals from fluorescent transgenic proteins and be used to quantify protein expression levels. This protocol describes a broadly applicable dissection, fixation, and immunostaining approach inDrosophilalarvae.
Use of DeTerm for AutomatedDrosophilaDendrite Arbor Terminal Counts
Authors: Kimura Minami , Tann Jason Y. , Wilkes Oliver R. , Xu Fangke , Skibbe Henrik , Moore Adrian W.
Published: May 2024

Neurons have a complex dendritic architecture that governs information flow through a circuit. Manual quantification of dendritic arbor morphometrics is time-consuming and can be inaccurate. Automated quantification systems such as DeTerm help to overcome these limitations. DeTerm is a software tool that automatically recognizes dendrite branch terminals with high precision. It uses an artificial neural network to label the terminals, count them, and provide each terminal's positional data. DeTerm can recognize the dendritic terminals ofDrosophiladendritic arborization (da) neurons, and it can also examine other types of neurons, including mouse Purkinje cells. It is freely available and works on Mac, Windows, and Linux. Here, we describe the use of DeTerm.
Mosquito Larvae and Pupae Transport from the Field
Authors: Ayala Diego , Shepard John J. , Rose Noah H.
Published: May 2024

Laboratory study of field-collected mosquitoes can allow researchers to better understand the ways variation within and among mosquito populations shapes burdens of mosquito-borne disease. TheAnopheles gambiaecomplex comprises the most important vectors of malaria, but it can be challenging to keep in the laboratory. For some species of mosquitoes, especiallyAn. gambiae, it is very difficult to bring viable eggs into the laboratory. Instead, it is preferable to collect larvae or pupae and then transport them as carefully as possible back to the laboratory. This simple protocol allows a researcher to start new laboratory colonies from larvae or pupae collected from natural breeding sites or proceed directly to their planned experiments. The use of natural breeding sites provides additional reassurance that the resulting colonies are representative of natural populations.
Examining Cell-Specific Localization inAedes aegyptiTissues with Fluorescence In Situ Hybridization
Authors: Sajadi Farwa , Paluzzi Jean-Paul V.
Published: May 2024

Fluorescence in situ hybridization (FISH) is a macromolecular recognition tool that uses RNA or DNA fragments combined with fluorophore- or digoxigenin-coupled nucleotides as probes to examine transcript localization through the presence or absence of complementary sequences in fixed tissues or samples under a fluorescent microscope. FISH technology has been highly effective for mapping genes and constructing a visual map of animal genomes. Here, we describe the application of FISH technology in theAedes aegyptimosquito, where it is specifically used to localize receptor transcripts in gut tissues/organs. The methods presented highlight the synthesis of RNA probes and describe the 2-d process of incubating the tissues/organs with the RNA probes. We also describe tyramide signal amplification for improved signal detection.
Examining Protein Localization inAedes aegyptiCells, Tissues, and Organs: Whole-Mount Immunohistochemistry
Authors: Afifi Salwa , Sajadi Farwa , Paluzzi Jean-Paul V.
Published: May 2024

Immunohistochemistry (IHC) is a powerful technique used for visualizing cellular components and determining the presence and/or location of proteins or other macromolecules in tissue samples. The classical IHC process involves the detection of epitopes using a highly specific primary antibody. This is followed by a secondary antibody that is coupled to a reporter molecule or fluorophore and capable of binding to the primary antibody and allowing for protein immunodetection. Although IHC does not routinely provide quantitative results compared to an enzyme-linked immunoassay or western blotting, it permits the localization of the proteins in intact tissues. This protocol describes an IHC assay for whole-bodyAedes aegyptimosquito tissues that is used to detect small proteins, specifically neuropeptide hormones. This method is useful for protein detection in whole-mount preparations; however, cross-section IHC is recommended to determine if a protein is localized in the apical versus basolateral membrane of tissues/organs or to visualize immunological distribution in larger, more complex preparations.
Membrane Protein Fractionation and Analysis through Western Blot inAedes aegyptiMalpighian Tubules
Authors: Picinic Britney , Paluzzi Jean-Paul V.
Published: May 2024

Western blot analysis is a well-known and dependable technique used to quantify protein abundance in a wide variety of samples. A major consideration for running a successful western blot is ensuring that the protein to be analyzed is purified appropriately. For work with membrane-bound proteins, traditional methods of protein processing such as the use of high-frequency sonication and ultracentrifugation to separate proteins from the membrane are being replaced with less time-consuming approaches. The use of a membrane fractionation kit, which involves the separation of membrane proteins from soluble (cytosolic) proteins, is effective in allowing for the quantification and analysis of membrane-bound proteins. In this protocol, we describe use of the membrane fractionation kit to isolate membrane-bound proteins, followed by western blot analysis, to observe protein abundance. The protocol involves methods that require organ (or tissue) collection, followed by protein processing, and a 2-d western blot procedure.
Amplification and Identification of Vertebrate Host CytochromecOxidase Subunit I (COI) DNA Barcoding Templates from Mosquito Blood Meals
Authors: Reeves Lawrence E. , Burkett-Cadena Nathan D.
Published: May 2024

Mosquitoes take blood meals from a diverse range of host animals and their host associations vary by species. Characterizing these associations is an important element of the transmission dynamics of mosquito-vectored pathogens. To characterize mosquito host associations, various molecular techniques have been developed, which are collectively referred to as blood meal analysis. DNA barcoding has diverse biological applications and is well-suited to mosquito blood meal analysis. The standard DNA barcoding marker for animals is a 5′ fragment of the cytochromecoxidase I (COI) gene. A major advantage of this marker is its taxonomic coverage in DNA sequence reference databases, making it feasible to identify a wider range of mosquito host species than with any other gene. However, theCOIgene contains high sequence variation at potential priming sites between vertebrate orders. Coupled with the need for primer sequences to be mismatched with mosquito priming sites so that annealing to mosquito DNA is inhibited, it can be difficult to design primers suitable for blood meal analysis applications. Several primers are available that perform well in mosquito blood meal analysis, annealing to priming sites for most vertebrate host taxa, but not to those of mosquitoes. Because priming site sequence variation among vertebrate taxa can cause amplification to fail, a hierarchical approach to DNA barcoding-based blood meal analysis can be applied. In such an approach, no single primer set is expected to be effective for 100% of potential host species. If amplification fails in the initial reaction, a subsequent reaction is attempted with primers that anneal to different priming sites, and so on, until amplification is successful.
Selecting Transgenic Mosquito Larvae with Puromycin
Authors: Marois Eric
Published: May 2024

The selection of mosquito transgenic larvae using a nonfluorescent approach can be advantageous to reserve fluorophores for downstream applications, such as immunostaining or for the study of promoter activity by cloning a fluorescence reporter gene under the control of that promoter. We previously reported that puromycin selection is efficient in transgenicAnopheles gambiaeorAnopheles coluzziilarvae expressing anOpIE2-pacselection marker. A concentration of puromycin of >10 µg/mL is lethal forAnopheleslarvae, unless they carry the resistance gene, conferring them resistance to puromycin concentrations of 25–80 µg/mL. A drawback of this fully dominant selection marker is that, unlike with fluorescence markers, homozygous transgenics cannot be distinguished from heterozygotes. Here, we outline the procedure for selecting puromycin-resistant transgenicAnopheleslarvae.
Whole-Mount Immunofluorescent Labeling of the Mosquito Central Nervous System
Authors: Younger Meg A.
Published: May 2024

Mosquito-borne disease is a major global public health issue. One path toward the development of evidence-based strategies to limit mosquito biting is the study of the mosquito nervous system—in particular, the sensory systems that drive biting behavior. The central nervous system of insects consists of the brain and the ventral nerve cord. Here, we describe a protocol for dissecting, immunofluorescent labeling, and imaging both of these structures in the mosquito. This protocol was optimized forAedes aegyptiand works well onAnopheles gambiaetissue. It has not been tested in other mosquito species, but we anticipate that it would work on a range of mosquitoes, and, if not, our protocol will provide a starting point from which to optimize. Notably, a limited number of antibodies cross-react withAe. aegyptiproteins. This protocol is intended for use with validated antibodies and can also be used to test new antibodies as they are generated. It has been successfully used to visualize protein tags, such as green fluorescent protein, that have been introduced into the mosquito to amplify or detect their presence.
Validating Single-Guide RNA forAedes aegyptiGene Editing
Authors: Lo Ivan Hok Yin , Matthews Benjamin J.
Published: May 2024

Creating transgenic mosquitoes allows for mechanistic studies of basic mosquito biology and the development of novel vector control strategies. CRISPR–Cas9 gene editing has revolutionized gene editing, including in mosquitoes. This protocol details part of the gene editing process ofAedes aegyptimosquitoes via CRISPR–Cas9, through testing and validating single-guide RNAs (sgRNAs). Gene editing activity varies depending on the sequence of sgRNAs used, so validation of sgRNA activity should be done before large-scale generation of mutants or transgenics. sgRNA is designed using online tools and synthesized in <1 h. Once mutants or transgenics are generated via embryo microinjection, sgRNA activity is validated by quick genotyping polymerase chain reaction (PCR) and DNA sequencing.
Measuring Oviposition Preference inAedes aegyptiMosquitoes
Authors: Mohammed Yunusa Garba , Tochor Nicholas Kristoff , Matthews Benjamin J.
Published: May 2024

The female mosquito is endowed with the responsibility of gene transfer from one generation to another through careful delivery of her eggs to an appropriate environment. To guarantee a successful reproduction process, female mosquitoes use an evaluative approach to select an oviposition site that will ensure that their progeny successfully hatch and develop from larvae to pupa to adult. Specific conditions must be met during oviposition site selection before gravid females deposit eggs, including but not limited to appropriate temperature, salinity, and pH; the presence of nutrition; and a low risk of predation. Mosquito species exhibit a remarkable diversity in their oviposition site selection behaviors. For instance,Aedes aegypti, which is a vector for Zika, dengue, yellow fever, and chikungunya viruses, prefers to lay its eggs in natural depressions (e.g., tree holes) or artificial containers. In contrast, other mosquito species, likeAnopheles, gravitate toward more open water bodies. Understanding the oviposition preference behavior of theAe. aegyptimosquito will facilitate the development of surveillance and control efforts to hinder the successful progression of this vector. Here, we present information on some of the known behaviors and preferences ofAe. aegyptiduring oviposition site selection, techniques to measure these preferences, and some open questions.
Design and Validation of Guide RNAs for CRISPR–Cas9 Genome Editing in Mosquitoes
Authors: Lo Ivan Hok Yin , Matthews Benjamin J.
Published: May 2024

CRISPR–Cas9 has revolutionized gene editing for traditional and nontraditional model organisms alike. This tool has opened the door to new mechanistic studies of basic mosquito biology as well as the development of novel vector control strategies based on CRISPR–Cas9, including gene drives that spread genetic elements in the population. Although the promise of the specificity, flexibility, and ease of deployment CRISPR is real, its implementation still requires empirical optimization for each new species of interest, as well as to each genomic target within a given species. Here, we provide an overview of designing and testing single-guide RNAs for the use of CRISPR-based gene editing tools.
Mapping Transcript Cell-Specific Localization and Protein Subcellular Localization in the Adult MosquitoAedes aegypti
Authors: Sajadi Farwa , Afifi Salwa , Picinic Britney , Paluzzi Jean-Paul V.
Published: May 2024

This introduction reviews techniques used to examine the distribution and expression of gene transcripts and proteins in a variety of tissues/organs in the medically important global disease vector mosquito,Aedes aegypti. Specifically, these methods allow the detection of cell-specific transcript expression by fluorescent in situ hybridization; facilitate immunohistochemical mapping of a protein of interest in whole-mount small tissue/organ samples; examine the subcellular localization of proteins, such as membrane transporters, through sectioning of paraffin-embedded tissue/organ samples; and finally, enable the efficient separation of cytosolic and membrane proteins for western blot analysis without the need for specialized equipment (e.g., ultracentrifuge) in the mosquitoAe. aegypti. Such techniques are useful to help answer fundamental questions in mosquito scientific research including (but not limited to) the identification of specific cells in an organ responsible for expressing a receptor of particular interest and necessary for eliciting a response to exogenous signals, including hormones. Moreover, changes in the subcellular localization of specific targets of interest can be assessed both qualitatively and quantitatively, providing insight into transient or long-term physiologically relevant regulation necessary for activity under experimental treatments or varied internal (e.g., development) or external (e.g., environmental stress) factors that might be normally experienced by the organism.
Study of Dendrite Differentiation UsingDrosophilaDendritic Arborization Neurons
Authors: Tann Jason Y. , Xu Fangke , Kimura Minami , Wilkes Oliver R. , Yoong Li-Foong , Skibbe Henrik , Moore Adrian W.
Published: May 2024

Neurons receive, process, and integrate inputs. These operations are organized by dendrite arbor morphology, and the dendritic arborization (da) neurons of theDrosophilaperipheral sensory nervous system are an excellent experimental model for examining the differentiation processes that build and shape the dendrite arbor. Studies in da neurons are enabled by a wealth of fly genetic tools that allow targeted neuron manipulation and labeling of the neuron's cytoskeletal or organellar components. Moreover, as da neuron dendrite arbors cover the body wall, they are highly accessible for live imaging analysis of arbor patterning. Here, we outline the structure and function of different da neuron types and give examples of how they are used to elucidate central mechanisms of dendritic arbor formation.
Cell Biology Techniques for StudyingDrosophilaPeripheral Glial Cells
Authors: Clayworth Katherine , Gilbert Mary , Auld Vanessa
Published: May 2024

Glial cells are essential for the proper development and functioning of the peripheral nervous system (PNS). The ability to study the biology of glial cells is therefore critical for our ability to understand PNS biology and address PNS maladies. The genetic and proteomic pathways underlying vertebrate peripheral glial biology are understandably complex, with many layers of redundancy making it sometimes difficult to study certain facets of PNS biology. Fortunately, many aspects of vertebrate peripheral glial biology are conserved with those of the fruit fly,Drosophila melanogaster. With simple and powerful genetic tools and fast generation times,Drosophilapresents an accessible and versatile model for studying the biology of peripheral glia. We introduce here three techniques for studying the cell biology of peripheral glia ofDrosophilathird-instar larvae. With fine dissection tools and common laboratory reagents, third-instar larvae can be dissected, with extraneous tissues removed, revealing the central nervous system (CNS) and PNS to be processed using a standard immunolabeling protocol. To improve the resolution of peripheral nerves in thez-plane, we describe a cryosectioning method to achieve 10- to 20-µm thick coronal sections of whole larvae, which can then be immunolabeled using a modified version of standard immunolabeling techniques. Finally, we describe a proximity ligation assay (PLA) for detecting close proximity between two proteins—thus inferring protein interaction—in vivo in third-instar larvae. These methods, further described in our associated protocols, can be used to improve our understanding ofDrosophilaperipheral glia biology, and thus our understanding of PNS biology.
Introduction to Techniques Used to Study Mosquito Neuroanatomy and Neural Circuitry
Authors: Guerina Florence V. , Patkar Ameya P. , Younger Meg A.
Published: May 2024

Mosquitoes transmit deadly pathogens from person to person as they obtain the blood meal that is essential for their life cycle. Female mosquitoes of many species are unable to reproduce without consuming protein that they obtain from blood. This developmental stage makes them highly efficient disease vectors of deadly pathogens. They can transmit pathogens between members of the same species and different species that can provide a route for evolving zoonotic viruses to jump from animals to humans. One possible way to develop novel strategies to combat pathogen transmission by mosquitoes is to study the sensory systems that drive mosquito reproductive behaviors, in particular the neural architecture and circuits of mosquito sensory afferent neurons, the central circuits that process sensory information, and the downstream circuits that drive reproductive behaviors. The study of mosquito neuroanatomy and circuitry also benefits basic neuroscience, allowing for comparative neuroanatomy in insect species, which has great value in the current model species-heavy landscape of neuroscience. Here, we introduce two important techniques that are used to study neuroanatomy and neural circuitry—namely, immunofluorescent labeling and neural tracing. We describe how to apply these approaches to study mosquito neuroanatomy and describe considerations for researchers using the techniques.
Heralded initialization of charge state and optical-transition frequency of diamond tin-vacancy centers
Authors: Brevoord Julia M. , De Santis Lorenzo , Yamamoto Takashi , Pasini Matteo , Codreanu Nina , Turan Tim , Beukers Hans K.C. , Waas Christopher , Hanson Ronald
Published: May 2024

Diamond tin-vacancy centers have emerged as a promising platform for quantum information science and technology. A key challenge for their use in more-complex quantum experiments and scalable applications is the ability to prepare the center in the desired charge state with the optical transition at a predefined frequency. Here we report on heralding such successful preparation using a combination of laser excitation, photon detection, and real-time logic. We first show that fluorescence photon counts collected during an optimized resonant probe pulse strongly correlate with the subsequent charge state and optical-transition frequency, enabling real-time heralding of the desired state through threshold photon counting. We then implement and apply this heralding technique to photoluminescence-excitation measurements, coherent optical driving, and an optical Ramsey experiment, finding strongly increased optical coherence with increasing threshold. Finally, we demonstrate that the prepared optical frequency follows the probe laser across the inhomogeneous linewidth, enabling tuning of the transition frequency over multiple homogeneous linewidths.

Published by the American Physical Society
2024
Designing moiré patterns by strain
Authors: Escudero Federico , Sinner Andreas , Zhan Zhen , Pantaleón Pierre A. , Guinea Francisco
Published: May 2024

Experiments conducted on two-dimensional twisted materials have revealed a plethora of moiré patterns with different forms and shapes. The formation of these patterns is usually attributed to the presence of small strains in the samples, which typically arise during their fabrication. In this paper we find that the superlattice structure of such systems actually depends crucially on the interplay between twist and strain. For systems composed of honeycomb lattices, we show that this can lead to the formation of practically any moiré geometry, even if each lattice is only slightly distorted. As a result, we show that under strain the moiré Brillouin zone is not a stretched irregular hexagon, but rather a primitive cell that changes according to the geometry of the strained moiré vectors. We identify the conditions for the formation of hexagonal moiré patterns arising solely due to shear or biaxial strain, thus opening the possibility of engineering moiré patterns solely by strain. Moreover, we study the electronic properties in such moiré patterns and find that the strain tends to suppress the formation of the flat moiré bands, even in the strain-induced hexagonal patterns analogous to those obtained by the twist only. Our paper explains the plethora of moiré patterns observed in experiments, and provides a solid theoretical foundation from which one can design moiré patterns by strain.

Published by the American Physical Society
2024
Breakdown of steady-state superradiance in extended driven atomic arrays
Authors: Ostermann Stefan , Rubies-Bigorda Oriol , Zhang Victoria , Yelin Susanne F.
Published: May 2024

Recent advances in generating well controlled dense arrangements of individual atoms in free space have generated interest in understanding how the extended nature of these systems influences superradiance phenomena. Here, we provide an in-depth analysis on how space-dependent light shifts and decay rates induced by dipole-dipole interactions modify the steady-state properties of coherently driven arrays of quantum emitters. We characterize the steady-state phase diagram, with particular focus on the radiative properties in the steady state. Interestingly, we find that diverging from the well-established Dicke paradigm of equal all-to-all interactions significantly modifies the emission properties. In particular, the prominent quadratic scaling of the radiated light intensity with particle number in the steady state—a hallmark of steady-state Dicke superradiance—is entirely suppressed, resulting in only linear scaling with particle number. We show that this breakdown of steady-state superradiance occurs due to the emergence of additional dissipation channels that populate not only superradiant states but also subradiant ones. The additional contribution of subradiant dark states in the dynamics leads to a divergence in the time scales needed to achieve steady states. Building on this, we further show that measurements taken at finite times for extended atom ensembles reveal properties closely mirroring the idealized Dicke scenario.

Published by the American Physical Society
2024