Site-specific genetic and functional signatures of aortic endothelial cells at aneurysm predilection sites in healthy and AngII ApoE-/- mice.

Aortic aneurysm is characterized by a pathological dilation at specific predilection sites of the vessel and potentially results in life-threatening vascular rupture. Herein, we established a modified "Häutchen method" for the local isolation of endothelial cells (ECs) from mouse aorta to analyze their spatial heterogeneity and potential role in site-specific disease development. When we compared ECs from aneurysm predilection sites of healthy mice with adjacent control segments we found regulation of genes related to extracellular matrix remodeling, angiogenesis and inflammation, all pathways playing a critical role in aneurysm development. We also detected enhanced cortical stiffness of the endothelium at these sites. Gene expression of ECs from aneurysms of the AngII ApoE-/- model when compared to sham animals mimicked expression patterns from predilection sites of healthy animals. Thus, this work highlights a striking genetic and functional regional heterogeneity in aortic ECs of healthy mice, which defines the location of aortic aneurysm formation in disease.

Insights from Murine Studies on the Site Specificity of Atherosclerosis.

Atherosclerosis is an inflammatory reaction that develops at specific regions within the artery wall and at specific sites of the arterial tree over a varying time frame in response to a variety of risk factors. The mechanisms that account for the interaction of systemic factors and atherosclerosis-susceptible regions of the arterial tree to mediate this site-specific development of atherosclerosis are not clear. The dynamics of blood flow has a major influence on where in the arterial tree atherosclerosis develops, priming the site for interactions with atherosclerotic risk factors and inducing cellular and molecular participants in atherogenesis. But how this accounts for lesion development at various locations along the vascular tree across differing time frames still requires additional study. Currently, murine models are favored for the experimental study of atherogenesis and provide the most insight into the mechanisms that may contribute to the development of atherosclerosis. Based largely on these studies, in this review, we discuss the role of hemodynamic shear stress, SR-B1, and other factors that may contribute to the site-specific development of atherosclerosis.

An Introductory Guide to Protease Sensitive Linker Design Using Matrix Metalloproteinase 13 as an Example.

Proteases play a crucial role, not only in physiological, but also in pathological processes, such as cancer, inflammation, arthritis, Alzheimer's, and infections, to name but a few. Their ability to cleave peptides can be harnessed for a broad range of biotechnological purposes. To do this efficiently, it is essential to find an amino acid sequence that meets the necessary requirements, including interdependent factors like specificity, selectivity, cleavage kinetics, or synthetic accessibility. Cleavage sequences from natural substrates of the protease may not be optimal in terms of specificity and selectivity, which is why these frequently require arduous and sometimes unsuccessful optimization such as by iterative exchange of single amino acids. Hence, here we describe the systematic design of protease sensitive linkers (PSLs)─peptide sequences specifically cleaved by a target protease─guided by the mass spectrometry based determination of target protease specific cleavage sites from a proteome-based peptide library. It includes a procedure for identifying bespoke PSL sequences, their optimization, synthesis, and validation and introduces a program that can indicate potential cleavage sites by hundreds of enzymes in any arbitrary amino acid sequence. Thereby, we provide an introduction to PSL design, illustrated by the example of matrix metalloproteinase 13 (MMP13). This introduction can serve as a guide and help to greatly accelerate the development and use of protease-sensitive linkers in diverse applications.

The microneme adhesive repeat domain of MIC3 protein determined the site specificity of Eimeria acervulina, Eimeria maxima, and Eimeria mitis.

Understanding the determinants of host and tissue tropisms among parasites of veterinary and medical importance has long posed a substantial challenge. Among the seven species of Eimeria known to parasitize the chicken intestine, a wide variation in tissue tropisms has been observed. Prior research suggested that microneme protein (MIC) composed of microneme adhesive repeat (MAR) domain responsible for initial host cell recognition and attachment likely dictated the tissue tropism of Eimeria parasites. This study aimed to explore the roles of MICs and their associated MARs in conferring site-specific development of E. acervuline, E. maxima, and E. mitis within the host. Immunofluorescence assays revealed that MIC3 of E. acervuline (EaMIC3), MIC3 of E. maxima (EmMIC3), MIC3 of E. mitis (EmiMIC3), MAR3 of EaMIC3 (EaMIC3-MAR3), MAR2 of EmMIC3 (EmMIC3-MAR2), and MAR4 of EmiMIC3 (EmiMIC3-MAR4), exhibited binding capabilities to the specific intestinal tract where these parasites infect. In contrast, the invasion of sporozoites into host intestinal cells could be significantly inhibited by antibodies targeting EaMIC3, EmMIC3, EmiMIC3, EaMIC3-MAR3, EmMIC3-MAR2, and EmiMIC3-MAR4. Substitution experiments involving MAR domains highlighted the crucial roles of EaMIC3-MAR3, EmMIC3-MAR2, and EmiMIC3-MAR4 in governing interactions with host ligands. Furthermore, animal experiments substantiated the significant contribution of EmiMIC3, EmiMIC3-MAR4, and their polyclonal antibodies in conferring protective immunity to Eimeria-affiliated birds. In summary, EaMIC3, EmMIC3, and EmiMIC3 are the underlying factors behind the diverse tissue tropisms exhibited by E. acervuline, E. maxima, and E. mitis, and EaMIC3-MAR3, EmMIC3-MAR2, and EmiMIC3-MAR4 are the major determinants of MIC-mediated tissue tropism of each parasite. The results illuminated the molecular basis of the modes of action of Eimeria MICs, thereby facilitating an understanding and rationalization of the marked differences in tissue tropisms among E. acervuline, E. maxima, and E. mitis.

Bisphosphonates and osteonecrosis of the jaws: Clinical and forensic aspects.

OBJECTIVE: This manuscript aims to provide a comprehensive review of the current knowledge in the pathophysiology, diagnosis, prevention, and other relevant clinical and forensic aspects of a potentially severe complication known as medication-related osteonecrosis of the jaw (MRONJ) while synthesizing state-of-the-art information on bisphosphonates and introducing a possible differential diagnosis. DESIGN: An extensive search was conducted in PubMed (U.S. National Library of Medicine) without a time or language constraint, focusing on the epidemiology, pathophysiology, risk factors, site specificity, signs and symptoms, differential diagnosis, prevention, and forensic aspects of MRONJ. All types of original articles, reviews, case reports, short communications, opinion articles, guidelines, and letters to editors were considered to produce a complete review on this subject. RESULTS: MRONJ prevention relies on a multidisciplinary approach and is critical since truly effective treatments are lacking. This therapeutic challenge is partly due to uncertainty regarding this condition's pathophysiology. Differential diagnosis of osteonecrosis of the jaws associated with krokodil abuse, one of the most dangerous and homemade psychoactive illicit substances, should be considered. CONCLUSIONS: Further research into the etiology and site specificity of MRONJ is encouraged, aiming to develop novel treatment prospects. Indeed, comprehending this would allow for increased efficacy and therapeutic options while emphasizing the importance of prevention. In addition, we advocate for greater consensus among the various societies regarding MRONJ's treatment and management.

Protein Modelling Highlighted Key Catalytic Sites Involved in Position-Specific Glycosylation of Isoflavonoids.

Uridine diphosphate glycosyltransferases (UGTs) are known for promiscuity towards sugar acceptors, a valuable characteristic for host plants but not desirable for heterologous biosynthesis. UGTs characterized for the O-glycosylation of isoflavonoids have shown a variable efficiency, substrate preference, and OH site specificity. Thus, 22 UGTs with reported isoflavonoid O-glycosylation activity were analyzed and ranked for OH site specificity and catalysis efficiency. Multiple-sequence alignment (MSA) showed a 33.2% pairwise identity and 4.5% identical sites among selected UGTs. MSA and phylogenetic analysis highlighted a comparatively higher amino acid substitution rate in the N-terminal domain that likely led to a higher specificity for isoflavonoids. Based on the docking score, OH site specificity, and physical and chemical features of active sites, selected UGTs were divided into three groups. A significantly high pairwise identity (67.4%) and identical sites (31.7%) were seen for group 1 UGTs. The structural and chemical composition of active sites highlighted key amino acids that likely define substrate preference, OH site specificity, and glycosylation efficiency towards selected (iso)flavonoids. In conclusion, physical and chemical parameters of active sites likely control the position-specific glycosylation of isoflavonoids. The present study will help the heterologous biosynthesis of glycosylated isoflavonoids and protein engineering efforts to improve the substrate and site specificity of UGTs.

Site-specific growth of gold nanoparticles on Bismuth Selenide hexagonal nanoplates.

Highly site-specific growth of gold nanoparticles (AuNPs) on Bismuth Selenide (Bi2Se3) hexagonal nanoplates was achieved by fine-tuning the growth kinetics of Au through controlling the coordination number of the Au ion in MBIA-Au3+ complex. With increasing concentration of MBIA, the increased amount and the coordination number of the MBIA-Au3+ complex results in the decrease of the reduction rate of Au. The slowed growth kinetics of Au allowed the recognition of the sites with different surface energy on the anisotropic Bi2Se3 hexagonal nanoplates. As a result, the site-specific growth of AuNPs at the corner, the edge, and the surface of the Bi2Se3 nanoplates were successfully achieved. This way of growth kinetic control was proven to be effective in constructing well-defined heterostructures with precise site-specificity and high purity of the product. This is helpful for the rational design and controlled synthesis of sophisticated hybrid nanostructures and would eventually promote their applications in various fields.

Comparative genomics and phenotypic studies to determine site-specificity of Escherichia coli in the lower gastrointestinal tract of humans.

Escherichia coli (E. coli) is an important commensal in the human gut; however, it is unknown whether strains show site-specificity in the lower gut. To investigate this, we assessed genotypic and phenotypic differences in 37 clone pairs (two strains with very similar multiple locus variable-number-tandem-repeat analysis [MLVA] profiles) of E. coli isolated from mucosal biopsies of two different gut locations (terminal ileum and rectum). The clone pairs varied at the genomic level; single nucleotide polymorphisms (SNPs) were common, multiple nucleotide polymorphisms (MNPs) were observed but less common, and few indels (insertions and deletions) were detected. The variation was higher in clone pairs that are associated with non-human-associated sequence types (ST) compared to human-associated STs, such as ST95, ST131, and ST73. No gene(s) with non-synonymous mutations were found to be commonly associated with either the terminal ileum or the rectal strains. At the phenotypic level, we identified the metabolic signatures for some STs. Rectum strains of some STs showed consistently higher metabolic activity with particular carbon sources. Clone pairs belonging to specific STs showed distinct growth patterns under different pH conditions. Overall, this study showed that E. coli may exhibit genomic and phenotypic variability at different locations in the gut. Although genomics did not reveal significant information suggesting the site-specificity of strains, some phenotypic studies have suggested that strains may display site-specificity in the lower gut. These results provide insights into the nature and adaptation of E. coli in the lower gut of humans. To the best of our knowledge, no study has investigated or demonstrated the site-specificity of commensal E. coli in the human gut.

Specificity for the correlation between the body surface and viscera in the pathological state of COPD: A prospective, controlled, and assessor-blinded trial.

Background: The association between the body surface and viscera remains obscure, but a better understanding of the body surface-viscera correlation will maximize its diagnostic and therapeutic values in clinical practice. Therefore, this study aimed to investigate the specificity of body surface-viscera correlation in the pathological state. Methods: The study subjects included 40 participants with chronic obstructive pulmonary disease (COPD) in the COPD group and 40 age-matched healthy participants in the healthy control group. Laser Doppler flowmetry, infrared thermography, and functional near-infrared spectroscopy were respectively adopted to measure 1) the perfusion unit (PU), 2) temperature, and 3) regional oxygen saturation (rSO2) of four specific sites distributed in the heart and lung meridians. These three outcome measures reflected the microcirculatory, thermal, and metabolic characteristics, respectively. Results: Regarding the microcirculatory and thermal characteristics of the body surface, the PU and temperature of specific sites on the body surface [i.e., Taiyuan (LU9) and Chize (LU5) in the lung meridian] in the COPD group were significantly increased compared with healthy controls (p < 0.05), whereas PU and temperature of other sites in the heart meridian [i.e., Shenmen (HT7) and Shaohai (HT3)] did not change significantly (p > 0.05). Regarding the metabolic characteristics, rSO2 of specific sites in the lung meridian [i.e., Taiyuan (LU9) and Chize (LU5)] and Shaohai (HT3) of the heart meridian in the COPD group was significantly decreased compared with healthy controls (p < 0.01), whereas rSO2 of Shenmen (HT7) in the heart meridian did not change significantly (p > 0.05). Conclusion: In the disease state of COPD, the microcirculatory, thermal, and metabolic characteristics of specific sites on the body surface in the lung meridian generally manifest more significant changes than those in the heart meridian, thereby supporting relative specificity for the body surface-viscera correlation in the pathological state.

Assaying for Arginyltransferase Activity and Specificity by Peptide Arrays.

Here, we describe arginylation assays performed on peptide arrays immobilized on cellulose membranes via chemical synthesis. In this assay, it is possible to simultaneously compare arginylation activity on hundreds of peptide substrates to analyze the specificity of arginyltransferase ATE1 toward its target site(s) and the amino acid sequence context. This assay was successfully employed in prior studies to dissect the arginylation consensus site and enable predictions of arginylated proteins encoded in eukaryotic genomes.

A model study on the site-specificity of (-)-epicatechin-induced reactions in ß-lactoglobulin by high-resolution mass spectrometry in combination with bioinformatics.

Polyphenol-protein reactions in model solutions of ß-lactoglobulin (ß-LG) incubated with (-)-epicatechin at 37 °C and 60 °C were monitored by microLC-timsTOF Pro-MS/MS combined with bioinformatics strategies. The addition of (-)-epicatechin to the model solutions resulted in changes in tryptic peptide profiles. Covalent bond formation between (-)-epicatechin o-quinones and ß-LG was identified for the residues S27, S30, K60, C66, K69, and C160, with C160 being the predominant binding site. Furthermore, the incubation of ß-LG with (-)-epicatechin significantly promoted oxidation, especially for the residues M7 and M24. The reaction of monomeric (-)-epicatechino-quinone at C160 was also identified in the milk chocolate sample. The adaptation of this study by extending the scope of the reaction products offers significant potential for comprehensive food profiling strategies.

Collision of Commonality and Personalization: Better Understanding of the Periosteum.

The periosteum is quite essential for bone repair. The excellent osteogenic properties of periosteal tissue make it a popular choice for accelerated osteogenesis in tissue engineering. With advances in research and technology, renewed attention has been paid to the periosteum. Recent studies have shown that the complexity of the periosteum is not only limited to histological features but also includes genetic and phenotypic features. In addition, the periosteum is proved to be quite site-specific in many ways. This brings challenges to the selection of periosteal donor sites. Limited understanding of the periosteum sets up barriers to developing optimal tissue regeneration strategies. A better understanding of periosteum could lead to better applications. Therefore, we reviewed the histological structure, gene expression, and function of the periosteum from both the commonality and personalization. It aims to discuss some obscure issues and untapped potential of periosteum and artificial periosteum in the application, where further theoretical research is needed. Overall, the site-specificity of the periosteum needs to be fully considered in future applications. However, significant further work is needed in relevant clinical trials to promote the further development of artificial periosteum.

Perivascular mechanical environment: A narrative review of the role of externally applied mechanical force in the pathogenesis of atherosclerosis.

Atherosclerosis is promoted by systemic factors, such as dyslipidemia, hypertension, diabetes, and smoking, which cause atherosclerosis in blood vessels throughout the body. However, atherosclerotic lesions are characterized by their frequent occurrence in specific vessels and sites. Blood vessels are exposed to various mechanical forces related to blood pressure and flow. Although shear stress promotes the initiation and progression of atherosclerotic lesions, the pathogenesis of site specificity of atherosclerosis is not sufficiently explained by shear stress. We propose the concept of a perivascular mechanical environment (PVME). Compelling evidence suggests that site specificity in atherosclerotic lesions depends on a distinct local PVME. Atheroprone arteries, such as the coronary artery, are markedly affected by externally applied mechanical force (EMF), whereas atheroprotective arteries, such as the internal thoracic artery, are less affected. Recent studies have shown that the coronary artery is affected by cardiac muscle contraction, the carotid artery by the hyoid bone and the thyroid cartilage, and the abdominal aorta and lower extremity arteries by musculoskeletal motion. We speculate that the thoracic cage protects the internal thoracic artery from EMF owing to a favorable PVME. Furthermore, evidence suggests that plaque eccentricity is provided by EMF; plaques are frequently observed on an external force-applied side. In each vascular tree, site-specific characteristics of the PVME differ substantially, inducing individual atherogenicity. From the perspective of the mechanical environment, hemodynamic stress occurs in an inside-out manner, whereas EMF occurs in an outside-in manner. These inward and outward forces apply mechanical load individually, but interact synergistically. The concept of a PVME is a novel pathogenesis of atherosclerosis and also might be a pathogenesis of other arterial diseases.

Structural basis for cell type specific DNA binding of C/EBPß: The case of cell cycle inhibitor p15INK4b promoter.

C/EBPß is a key regulator of numerous cellular processes, but it can also contribute to tumorigenesis and viral diseases. It binds to specific DNA sequences (C/EBP sites) and interacts with other transcription factors to control expression of multiple eukaryotic genes in a tissue and cell-type dependent manner. A body of evidence has established that cell-type-specific regulatory information is contained in the local DNA sequence of the binding motif. In human epithelial cells, C/EBPß is an essential cofactor for TGFß signaling in the case of Smad2/3/4 and FoxO-dependent induction of the cell cycle inhibitor, p15INK4b. In the TGFß-responsive region 2 of the p15INK4b promoter, the Smad binding site is flanked by a C/EBP site, CTTAA•GAAAG, which differs from the canonical, palindromic ATTGC•GCAAT motif. The X-ray crystal structure of C/EBPß bound to the p15INK4b promoter fragment shows how GCGC-to-AAGA substitution generates changes in the intermolecular interactions in the protein-DNA interface that enhances C/EBPß binding specificity, limits possible epigenetic regulation of the promoter, and generates a DNA element with a unique pattern of methyl groups in the major groove. Significantly, CT/GA dinucleotides located at the 5'ends of the double stranded element maintain local narrowing of the DNA minor groove width that is necessary for DNA recognition. Our results suggest that C/EBPß would accept all forms of modified cytosine in the context of the CpT site. This contrasts with the effect on the consensus motif, where C/EBPß binding is modestly increased by cytosine methylation, but substantially decreased by hydroxymethylation.

SPIONs Conjugate Supported Anticancer Drug Doxorubicin's Delivery: Current Status, Challenges, and Prospects.

Considerable efforts have been directed towards development of nano-structured carriers to overcome the limitations of anticancer drug, doxorubicin's, delivery to various cancer sites. The drug's severe toxicity to cardio and hepatic systems, low therapeutic outcomes, inappropriate dose-demands, metastatic and general resistance, together with non-selectivity of the drug have led to the development of superparamagnetic iron oxide nanoparticles (SPIONs)-based drug delivery modules. Nano-scale polymeric co-encapsulation of the drug, doxorubicin, with SPIONs, the SPIONs surface end-groups' cappings with small molecular entities, as well as structural modifications of the SPIONs' surface-located functional end-groups, to attach the doxorubicin, have been achieved through chemical bonding by conjugation and cross-linking of natural and synthetic polymers, attachments of SPIONs made directly to the non-polymeric entities, and attachments made through mediation of molecular-spacer as well as non-spacer mediated attachments of several types of chemical entities, together with the physico-chemical bondings of the moieties, e.g., peptides, proteins, antibodies, antigens, aptamers, glycoproteins, and enzymes, etc. to the SPIONs which are capable of targeting multiple kinds of cancerous sites, have provided stable and functional SPIONs-based nano-carriers suitable for the systemic, and in vitro deliveries, together with being suitable for other biomedical/biotechnical applications. Together with the SPIONs inherent properties, and ability to respond to magnetic resonance, fluorescence-directed, dual-module, and molecular-level tumor imaging; as well as multi-modular cancer cell targeting; magnetic-field-inducible drug-elution capacity, and the SPIONs' magnetometry-led feasibility to reach cancer action sites have made sensing, imaging, and drug and other payloads deliveries to cancerous sites for cancer treatment a viable option. Innovations in the preparation of SPIONs-based delivery modules, as biocompatible carriers; development of delivery route modalities; approaches to enhancing their drug delivery-cum-bioavailability have explicitly established the SPIONs' versatility for oncological theranostics and imaging. The current review outlines the development of various SPIONs-based nano-carriers for targeted doxorubicin delivery to different cancer sites through multiple methods, modalities, and materials, wherein high-potential nano-structured platforms have been conceptualized, developed, and tested for, both, in vivo and in vitro conditions. The current state of the knowledge in this arena have provided definite dose-control, site-specificity, stability, transport feasibility, and effective onsite drug de-loading, however, with certain limitations, and these shortcomings have opened the field for further advancements by identifying the bottlenecks, suggestive and plausible remediation, as well as more clear directions for future development.

Medication-related osteonecrosis of the jaw: A literature review and update.

Since the initial description of medication-related osteonecrosis of the jaw (MRONJ) almost two decades ago, the potential pathophysiology and risk factors have been elaborated on in many investigations and guidelines. However, the definitive pathophysiology based on scientific evidence remains lacking. Consequently, the optimal clinical treatment and prevention strategies for MRONJ have not been established. Despite their different mechanisms of action, many drugs, including bisphosphonates, denosumab, angiogenesis inhibitors, and other medications, have been reported to be associated with MRONJ lesions in cancer and osteoporosis patients. Importantly, MRONJ occurs predominantly in the jawbones and other craniofacial regions, but not in the appendicular skeleton. In this up-to-date review, the currently available information and theories regarding MRONJ are presented from both clinical and basic science perspectives. The definition and epidemiology of MRONJ, triggering medication, and histopathology are comprehensively summarized. The immunopathology and the potential pathophysiology based on immune cells such as neutrophils, T and B cells, macrophages, dendritic cells, and natural killer cells are also discussed. In addition, antiangiogenesis, soft tissue toxicity, necrotic bone, osteocyte death, and single-nucleotide polymorphisms are examined. Moreover, other possible mechanisms of MRONJ development are considered based on the unique embryological characteristics, different cell behaviors between jawbones and appendicular skeleton, unique anatomical structures, and sustained bacterial exposure in the oral cavity as a basis for MRONJ site specificity. Based on the literature review, it was concluded that multiple factors may contribute to the development of MRONJ, although which one is the key player triggering MRONJ in the craniofacial region remains unknown.

Assessment of Compound-Specific Fatty Acid δ13C and δ2H Values to Track Fish Mobility in a Small Sub-alpine Catchment.

Methods for identifying origin, movement, and foraging areas of animals are essential for understanding ecosystem connectivity, nutrient flows, and other ecological processes. Telemetric methods can provide detailed spatial coverage but are limited to a minimum body size of specimen for tagging. In recent years, stable isotopes have been increasingly used to track animal migration by linking landscape isotope patterns into movement (isoscapes). However, compared to telemetric methods, the spatial resolution of bulk stable isotopes is low. Here, we examined a novel approach by evaluating the use of compound-specific hydrogen and carbon stable isotopes of fatty acids (δ2HFA and δ13CFA) from fish liver, muscle, brain, and eye tissues for identifying site specificity in a 254 km2 sub-alpine river catchment. We analyzed 208 fish (European bullhead, rainbow trout, and brown trout) collected in 2016 and 2018 at 15 different sites. δ13CFA values of these fish tissues correlated more among each other than those of δ2HFA values. Both δ2HFA and δ13CFA values showed tissue-dependent isotopic fractionation, while fish taxa had only small effects. The highest site specificity was for δ13CDHA values, while the δ2H isotopic difference between linoleic acid and alpha-linolenic acid resulted in the highest site specificity. Using linear discrimination analysis of FA isotope values, over 90% of fish could be assigned to their location of origin; however, the accuracy dropped to about 56% when isotope data from 2016 were used to predict the sites for samples collected in 2018, suggesting temporal shifts in site specificity of δ2HFA and δ13CFA. However, the predictive power of δ2HFA and δ13CFA over this time interval was still higher than site specificity of bulk tissue isotopes for a single time point. In summary, compound-specific isotope analysis of fatty acids may become a highly effective tool for assessing fine and large-scale movement and foraging areas of animals.

The Moxibustion-Induced Thermal Transport Effect Between the Heart and Lung Meridians With Infrared Thermography.

Objectives: By comparing the differences in the thermal transport effect between the heart and lung meridians induced by moxibustion, this study aimed to investigate the specificity of site-to-site associations on the body surface between different meridians. Methods: Eighty healthy participants were divided into the heart meridian intervention group and the lung meridian intervention group; moxibustion was performed at these two meridians, respectively. Baseline temperature and its change magnitude from baseline induced by moxibustion in 6 measuring sites of the heart and lung meridians were assessed by infrared thermography (IRT). Measuring sites included: Site 1 (Chize, LU5), Site 2 (midpoint of LU9 and LU5), Site 3 (Taiyuan, LU9), Site 4 (Shaohai, HT3), Site 5 (midpoint of HT7 and HT3), and Site 6 (Shenmen, HT7). Results: Forty participants (20 male and 20 female, 27.90 ± 0.52 years) were assigned to the heart meridian intervention group, and 40 participants (20 male and 20 female, 28.08 ± 0.54 years) were assigned to the lung meridian intervention group. In the lung meridian intervention group (moxibustion over LU5), the temperature of the distal sites in the lung meridian increased significantly at 5, 10, and 15 min compared with pre-moxibustion (P < 0.001). The temperature of Site 4 in the heart meridian, which was nearest to the moxibustion site, increased significantly compared with pre-moxibustion (P < 0.05), while the temperature in the distal sites of the heart meridian did not differ significantly during moxibustion. Regarding the comparison of temperature change magnitude from baseline (ΔT) between the two meridians, the ΔT of Site 2 in the lung meridian was significantly higher than Site 4 in the heart meridian at 5 and 10 min after moxibustion (P < 0.05), despite that Site 2 was more distal from the moxibustion site than Site 4. Similarly, the ΔT of Site 3 in the lung meridian was significantly higher than Site 5 and Site 6 in the heart meridian at 5, 10, and 15 min after moxibustion (P < 0.05). In the heart meridian invervention group, similar thermal transport effect between the two meridians was observed. The thermal transport effect of the distal sites along the heart meridian was more significant than that of the site closer to the moxibustion site but located in the lung meridian. Taken together, aforementioned results indicated that the moxibustion-induced thermal transport effect between the heart and lung meridians is generally more significant in the distal sites along the corresponding meridian than that in the closer sites of the other meridian. Conclusions: In the heart and lung meridians, the moxibustion-induced thermal transport effect is closely related to meridian routes, not just related to the absolute distance from the moxibustion site, thereby confirming the relative specificity of "site-to-site" associations on the body surface in these two meridians. Systematic Review Registration: https://clinicaltrials.gov/ct2/show/NCT05330403, identifier NCT05330403.

Site-specific nanobody-oligonucleotide conjugation for super-resolution imaging.

Camelid single-domain antibody fragments, also called nanobodies, constitute a class of binders that are small in size (~15 kDa) and possess antigen-binding properties similar to their antibody counterparts. Facile production of recombinant nanobodies in several microorganisms has made this class of binders attractive within the field of molecular imaging. Particularly, their use in super-resolution microscopy has improved the spatial resolution of molecular targets due to a smaller linkage error. In single-molecule localization microscopy techniques, the effective spatial resolution can be further enhanced by site-specific fluorescent labeling of nanobodies owing to a more homogeneous protein-to-fluorophore stoichiometry, reduced background staining and a known distance between dye and epitope. Here, we present a protocol for site-specific bioconjugation of DNA oligonucleotides to three distinct nanobodies expressed with an N- or C-terminal unnatural amino acid, 4-azido-L-phenylalanine (pAzF). Using copper-free click chemistry, the nanobody-oligonucleotide conjugation reactions were efficient and yielded highly pure bioconjugates. Target binding was retained in the bioconjugates, as demonstrated by bio-layer interferometry binding assays and the super-resolution microscopy technique, DNA points accumulation for imaging in nanoscale topography (PAINT). This method for site-specific protein-oligonucleotide conjugation can be further extended for applications within drug delivery and molecular targeting where site-specificity and stoichiometric control are required.

N-Glycoprofiling of immunoglobulin G and lactoferrin with site-specificity from goat milk using RP-UHPLC MS/MS.

Glycans present in glycoproteins are structurally diverse and contribute to the carbohydrate pool of the milk. Goat milk is a leading non-bovine milk source, wherein glycan diversity of several glycoproteins remains unexplored. Herein, site-specific N-glycoprofiling of two major glycoproteins - immunoglobulin G (IgG) and lactoferrin (Lf) from goat milk was performed through RP-UHPLC Q-Tof MS/MS approach. IgG revealed diverse complex glycans that were predominantly biantennary type with differential core fucosylation, bisecting GlcNAc, and mono/di- sialylation (NeuAc/NeuGc). The N-glycan repertoire of Lf at four sites indicated the range of high mannose, complex and hybrid types with varying abundances. High mannose glycans were specifically observed at N252NT and N564DT sites. Majorly complex glycans with fully sialylated were found at N387VT site. While N495QT site revealed complex and hybrid types with differential core fucosylation and sialylation. The glycan features observed in these glycoproteins would pave way for effective utilization as bioactive ingredients.