Are the European reference networks for rare diseases ready to embrace machine learning? A mixed-methods study.

BACKGROUND: The delay in diagnosis for rare disease (RD) patients is often longer than for patients with common diseases. Machine learning (ML) technologies have the potential to speed up and increase the precision of diagnosis in this population group. We aim to explore the expectations and experiences of the members of the European Reference Networks (ERNs) for RDs with those technologies and their potential for application. METHODS: We used a mixed-methods approach with an online survey followed by a focus group discussion. Our study targeted primarily medical professionals but also other individuals affiliated with any of the 24 ERNs. RESULTS: The online survey yielded 423 responses from ERN members. Participants reported a limited degree of knowledge of and experience with ML technologies. They considered improved diagnostic accuracy the most important potential benefit, closely followed by the synthesis of clinical information, and indicated the lack of training in these new technologies, which hinders adoption and implementation in routine care. Most respondents supported the option that ML should be an optional but recommended part of the diagnostic process for RDs. Most ERN members saw the use of ML limited to specialised units only in the next 5 years, where those technologies should be funded by public sources. Focus group discussions concluded that the potential of ML technologies is substantial and confirmed that the technologies will have an important impact on healthcare and RDs in particular. As ML technologies are not the core competency of health care professionals, participants deemed a close collaboration with developers necessary to ensure that results are valid and reliable. However, based on our results, we call for more research to understand other stakeholders' opinions and expectations, including the views of patient organisations. CONCLUSIONS: We found enthusiasm to implement and apply ML technologies, especially diagnostic tools in the field of RDs, despite the perceived lack of experience. Early dialogue and collaboration between health care professionals, developers, industry, policymakers, and patient associations seem to be crucial to building trust, improving performance, and ultimately increasing the willingness to accept diagnostics based on ML technologies.

Glucagon- and insulin-immunopositive endocrine cells in porcine extrahepatic bile ducts and gallbladder.

Introduction: Pancreatic ß-cells and α-cells have been found in the murine extrahepatic biliary ducts but not in the gallbladder. However, there has been no information reported in the specialized literature about the presence of glucagon- and insulin-expressing endocrine cells in porcine bile ducts and gallbladder. Aim: We aimed to perform an immunohistochemical study to identify glucagon- and insulin-positive cells and their distribution in the porcine extrahepatic biliary ducts and gallbladder. Method: The immunohistochemical method was used to detect the presence and distribution of glucagon- and insulin-positive endocrine cells in the common hepatic duct (ductus hepaticus communis), common bile duct (ductus choledochus), cystic duct (ductus cysticus), and gallbladder (vesica fellea) of male pigs. Chromogranin A was used as a typical marker for endocrine cells. Results: The density of chromogranin A-, glucagon- and insulin-positive cells per field was the largest in the common bile duct, followed by the common hepatic duct, cystic duct, and gallbladder. The three types of endocrine cells showed specific localization in the superficial and deep glands of the studied organs. Conclusion and clinical importance: The distribution of glucagon- and insulin-immunopositive endocrine cells in the porcine extrahepatic biliary tract was established for the first time as a new source of these hormones. The presence of α- and ß-cells in the epithelium of extrahepatic bile ducts can be applied in treatment of diabetes, taking into account the possibility to reprogram the biliary epithelium to mentioned pancreatic endocrine cell types.

Doxorubicin Changes the Spatial Organization of the Genome around Active Promoters.

In this study, we delve into the impact of genotoxic anticancer drug treatment on the chromatin structure of human cells, with a particular focus on the effects of doxorubicin. Using Hi-C, ChIP-seq, and RNA-seq, we explore the changes in chromatin architecture brought about by doxorubicin and ICRF193. Our results indicate that physiologically relevant doses of doxorubicin lead to a local reduction in Hi-C interactions in certain genomic regions that contain active promoters, with changes in chromatin architecture occurring independently of Top2 inhibition, cell cycle arrest, and differential gene expression. Inside the regions with decreased interactions, we detected redistribution of RAD21 around the peaks of H3K27 acetylation. Our study also revealed a common structural pattern in the regions with altered architecture, characterized by two large domains separated from each other. Additionally, doxorubicin was found to increase CTCF binding in H3K27 acetylated regions. Furthermore, we discovered that Top2-dependent chemotherapy causes changes in the distance decay of Hi-C contacts, which are driven by direct and indirect inhibitors. Our proposed model suggests that doxorubicin-induced DSBs cause cohesin redistribution, which leads to increased insulation on actively transcribed TAD boundaries. Our findings underscore the significant impact of genotoxic anticancer treatment on the chromatin structure of the human genome.

CYCLeR-a novel tool for the full isoform assembly and quantification of circRNAs.

Splicing is one key mechanism determining the state of any eukaryotic cell. Apart from linear splice variants, circular splice variants (circRNAs) can arise via non-canonical splicing involving a back-splice junction (BSJ). Most existing methods only identify circRNAs via the corresponding BSJ, but do not aim to estimate their full sequence identity or to identify different, alternatively spliced circular isoforms arising from the same BSJ. We here present CYCLeR, the first computational method for identifying the full sequence identity of new and alternatively spliced circRNAs and their abundances while simultaneously co-estimating the abundances of known linear splicing isoforms. We show that CYCLeR significantly outperforms existing methods in terms of F score and quantification of transcripts in simulated data. In a in a comparative study with long-read data, we also show the advantages of CYCLeR compared to existing methods. When analysing Drosophila melanogaster data, CYCLeR uncovers biological patterns of circRNA expression that other methods fail to observe.

Incorporation of the Dry Blossom Flour of Sambucus nigra L. in the Production of Sponge Cakes.

The present study aims to develop recipe compositions and technology for producing sponge cakes from wholemeal flour, partially replaced with a functional plant component dry blossom flour of Sambucus nigra L. Three designs of sponge cakes with 5, 10, and 15% content of flour of Sambucus nigra L. corrected up to 100% with whole-grain oat flour were studied. Their characteristics were compared with sponge cakes of 100% wheat flour/control. The obtained new products were characterized by reduced carbohydrates, increased content of dietary fiber, and preserved volume compared to the control. The physicochemical parameters of sponge cake and marshmallows with different concentrations of dry flowers of Sambucus nigra L. included in them differed from the control with lower water absorption, pH, and moisture, while having a higher relative mass and ash content and retaining the original size. Pathogenic microorganisms such as Escherichia coli, Salmonella sp., and Staphylococcus aureus, and common coliforms were not detected in the control and experimental samples when determining the microbiological parameters. Therefore, the developed formulations are an excellent alternative to wheat flour, significantly improving some nutritional characteristics such as smell, taste, dietary fiber, and lower carbohydrate content.

Lipid Nanoparticulate Drug Delivery Systems: Recent Advances in the Treatment of Skin Disorders.

The multifunctional role of the human skin is well known. It acts as a sensory and immune organ that protects the human body from harmful environmental impacts such as chemical, mechanical, and physical threats, reduces UV radiation effects, prevents moisture loss, and helps thermoregulation. In this regard, skin disorders related to skin integrity require adequate treatment. Lipid nanoparticles (LN) are recognized as promising drug delivery systems (DDS) in treating skin disorders. Solid lipid nanoparticles (SLN) together with nanostructured lipid carriers (NLC) exhibit excellent tolerability as these are produced from physiological and biodegradable lipids. Moreover, LN applied to the skin can improve stability, drug targeting, occlusion, penetration enhancement, and increased skin hydration compared with other drug nanocarriers. Furthermore, the features of LN can be enhanced by inclusion in suitable bases such as creams, ointments, gels (i.e., hydrogel, emulgel, bigel), lotions, etc. This review focuses on recent developments in lipid nanoparticle systems and their application to treating skin diseases. We point out and consider the reasons for their creation, pay attention to their advantages and disadvantages, list the main production techniques for obtaining them, and examine the place assigned to them in solving the problems caused by skin disorders.

Low-dose rivaroxaban plus aspirin in older patients with peripheral artery disease undergoing acute limb revascularization: insights from the VOYAGER PAD trial.

AIMS: In this secondary analysis of the VOYAGER trial, rivaroxaban 2.5 mg twice/day plus aspirin 100 mg/day was assessed in older adults. Advanced age is associated with elevated bleeding risk and unfavourable net benefit for dual antiplatelet therapy in chronic coronary artery disease. The risk-benefit of low-dose rivaroxaban in patients ≥75 years with peripheral artery disease (PAD) after lower extremity revascularization (LER) has not been described. METHODS AND RESULTS: The primary endpoint was a composite of acute limb ischaemia, major amputation, myocardial infarction, ischaemic stroke, or cardiovascular death. The principal safety outcome was thrombolysis in myocardial infarction (TIMI) major bleeding analysed by the pre-specified age cut-off of 75 years. Of 6564 patients randomized, 1330 (20%) were >75 years. Absolute 3-year Kaplan-Meier cumulative incidence rates for primary efficacy (23.4% vs. 19.0%) and safety (3.5% vs. 1.5%) endpoints were higher in elderly vs. non-elderly patients. Efficacy of rivaroxaban (P-interaction 0.83) and safety (P-interaction 0.38) was consistent irrespective of age. The combination of intracranial and fatal bleeding was not increased in patients >75 years (2 rivaroxaban vs. 8 placebo). Overall, benefits (absolute risk reduction 3.8%, number needed to treat 26 for the primary endpoint) exceeded risks (absolute risk increase 0.81%, number needed to harm 123 for TIMI major bleeding). CONCLUSION: Patients ≥75 years with PAD are at both heightened ischaemic and bleeding risk after LER. No excess harm with respect to major, intracranial or fatal bleeding was seen in older patients yet numerically greater absolute benefits were observed. This suggests that low-dose rivaroxaban combined with aspirin should be considered in PAD after LER regardless of age.

Natural Lipids as Structural Components of Solid Lipid Nanoparticles and Nanostructured Lipid Carriers for Topical Delivery.

BACKGROUND: Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) are useful drug delivery systems for dermal application. Thanks to their biocompatible and biodegradable profile, these carriers offer many advantages such as enhanced bioavailability, low toxicity, viable drug targeting and controlled release. SLN and NLC are composed of well-tolerated lipids, including natural fats and oils that are successfully used in the pharmaceutical and cosmetic dermal formulation. OBJECTIVE: This article presents an overview of the benefits of selecting natural fats and oils as structural components of SLN and NLC for topical application. METHODS: This review is based on data published over the past 20 years about the development of stable and nontoxic lipid nanoparticles with natural lipids. We shed light on the role of natural fats in skin restoration, as well as on the contributed penetration and occlusive properties of SLN and NLC. RESULTS: The deliberate selection of excipients (type and lipid ratio) influences the quality of the final dermal formulation. Natural lipids show good compatibility with different active molecules and are able to create stable lipid matrices that facilitate the biopharmaceutical properties of lipid nanoparticles. Patents involving natural fats and oils in SLN and NLC composition are listed, yet it is important to note that the approved marketed formulations are mainly cosmetic, not pharmaceutical, products. CONCLUSION: Natural lipids can enhance topical drug delivery by adding their ability of improving skin penetration and hydration to the permeation and occlusion properties of SLN and NLC.

Comparison of Vitreomacular Interface Changes in Myopic Foveoschisis and Idiopathic Epiretinal Membrane Foveoschisis.

PURPOSE: To compare characteristics of the vitreomacular interface (VMI) in myopic foveoschisis (mFS) and idiopathic epiretinal membrane foveoschisis (iERM-FS), and to correlate with optical coherence tomography and clinical data. DESIGN: Clinicopathologic study. METHODS: Epiretinal membrane and internal limiting membrane (ILM) specimens were removed from eyes with mFS (5 eyes) and iERM-FS (5 eyes). Harvested tissue was processed for immunocytochemistry and prepared by ultrathin series sectioning for transmission electron microscopy. Cell and collagen compositions were compared and correlated with clinical data. RESULTS: All eyes presented fibrocellular membranes irrespective if associated with mFS or iERM-FS. Cell and collagen types and distribution on the vitreal side of the ILM were similar in both groups, consistent with presence of tractional membranes on optical coherence tomography images. Immunostaining of all specimens were positive for glial cells, microglia, and hyalocytes. Electron microscopy revealed evidence of epiretinal cell multilayers with masses of vitreous collagen and signs of vitreous remodeling in both groups. Three eyes with mFS but none of the eyes with iERM-FS showed massive thinning of the ILM with prominent retinal undulations and presence of retinal nerve fiber layer fragments. CONCLUSION: Whereas fibrocellular components of premacular tissue in mFS are similar to iERM-FS, pathologic abnormalities of the ILM were exclusively present in high myopia. Although peeling of the ILM appears important to completely remove tractional components of the VMI, histopathologic findings emphasize the risk for retinal damage in these highly myopic eyes, indicating that individual preoperative assessment and modification of surgical techniques require further investigation.

Knocking out C9ORF72 Exacerbates Axonal Trafficking Defects Associated with Hexanucleotide Repeat Expansion and Reduces Levels of Heat Shock Proteins.

In amyotrophic lateral sclerosis (ALS) motor neurons (MNs) undergo dying-back, where the distal axon degenerates before the soma. The hexanucleotide repeat expansion (HRE) in C9ORF72 is the most common genetic cause of ALS, but the mechanism of pathogenesis is largely unknown with both gain- and loss-of-function mechanisms being proposed. To better understand C9ORF72-ALS pathogenesis, we generated isogenic induced pluripotent stem cells. MNs with HRE in C9ORF72 showed decreased axonal trafficking compared with gene corrected MNs. However, knocking out C9ORF72 did not recapitulate these changes in MNs from healthy controls, suggesting a gain-of-function mechanism. In contrast, knocking out C9ORF72 in MNs with HRE exacerbated axonal trafficking defects and increased apoptosis as well as decreased levels of HSP70 and HSP40, and inhibition of HSPs exacerbated ALS phenotypes in MNs with HRE. Therefore, we propose that the HRE in C9ORF72 induces ALS pathogenesis via a combination of gain- and loss-of-function mechanisms.

Endoscopic Interhemispheric Disconnection for Intractable Multifocal Epilepsy: Surgical Technique and Functional Neuroanatomy.

BACKGROUND: Callosotomy represents a palliative procedure for intractable multifocal epilepsy. The extent of callosotomy and the benefits of adding anterior and posterior commissurotomy are debated. OBJECTIVE: To describe a new technique of a purely endoscopic procedure to disconnect the corpus callosum, the anterior, posterior, and habenular commissures through the use of a single burr hole via a transfrontal transventricular route. METHODS: Our surgical series was retrospectively reviewed in terms of seizure control (Engel's class) and complication rate. Five cadaveric specimens were used to demonstrate the surgical anatomy of commissural fibers and third ventricle. RESULTS: The procedure may be divided into 3 steps: (1) endoscopic transventricular transforaminal anterior commissure disconnection; (2) disconnection of posterior and habenular commissures; and (3) total callosotomy. Fifty-seven patients were included in the analysis. A favorable outcome in terms of epilepsy control (Engel class 1 to 3) was found in 71.4% of patients undergoing callosotomy coupled with anterior, posterior, and habenular commissure disconnection against 53% of patients with isolated callosotomy (P = .26). Patients with drop attacks had better epilepsy outcome independently from the surgical procedure used. CONCLUSION: The full endoscopic callosotomy coupled with disconnection of anterior, posterior and habenular commissures is a safe alternative to treat multifocal refractory epilepsy. A gain in seizure outcome might be present in this cohort of patients treated with total interhemispheric disconnection when compared with isolated callosotomy. Larger studies are required to confirm these findings.

Arthritis in Two Patients With Partial Recombination Activating Gene Deficiency.

Autoimmunity is becoming an increasingly recognized complication in patients with primary immunodeficiencies (PIDs), including a variety of combined immune deficiencies such as Recombination Activating Gene (RAG) defects. The approach to treating autoimmunity in PID patients is complex, requiring a balance between immunosuppression and susceptibility to infection. Inflammatory arthritis is a feature of immune dysregulation in many PIDs, and the optimal treatment may differ from first line therapies that usually consist of disease-modifying anti rheumatic drugs (DMARDs). An example of mechanism-based therapy of arthritis in PID uses blockade of IL-6 signaling with tocilizumab for patients with STAT 3 gain-of-function (GOF) mutation and augmented IL-6 pathway. Herein, we describe two PID cases with arthritis who were found to have defects in RAG. One patient with refractory inflammatory arthritis experienced remarkable improvement in symptoms with tocilizumab therapy. Arthritis can be a clinical feature of immune dysregulation in RAG deficiency, and tocilizumab therapy has been suggested to have utility in treatment of arthritis in RAG deficiency.

Gas Mixing and Final Mixture Composition Control in Simple Geometry Micro-mixers via DSMC Analysis.

The mixing process of two pressure driven steady-state rarefied gas streams flowing between two parallel plates was investigated via DSMC (Direct Simulation Monte Carlo) for different combinations of gases. The distance from the inlet, where the associated relative density difference of each species is minimized and the associated mixture homogeneity is optimized, is the so-called mixing length. In general, gas mixing progressed very rapidly. The type of gas surface interaction was clearly the most important parameter affecting gas mixing. As the reflection became more specular, the mixing length significantly increased. The mixing lengths of the HS (hard sphere) and VHS (variable hard sphere) collision models were higher than those of the VSS (variable soft sphere) model, while the corresponding relative density differences were negligible. In addition, the molecular mass ratio of the two components had a minor effect on the mixing length and a more important effect on the relative density difference. The mixture became less homogenous as the molecular mass ratio reduced. Finally, varying the channel length and/or the wall temperature had a minor effect. Furthermore, it was proposed to control the output mixture composition by adding in the mixing zone, the so-called splitter, separating the downstream flow into two outlet mainstreams. Based on intensive simulation data with the splitter, simple approximate expressions were derived, capable of providing, once the desired outlet mixture composition was specified, the correct position of the splitter, without performing time consuming simulations. The mixing analysis performed and the proposed approach for controlling gas mixing may support corresponding experimental work, as well as the design of gas micro-mixers.

Transcriptional dynamics of microRNAs and their targets during Drosophila neurogenesis.

During Drosophila melanogaster embryogenesis, tight regulation of gene expression in time and space is required for the orderly emergence of specific cell types. While the general importance of microRNAs in regulating eukaryotic gene expression has been well-established, their role in early neurogenesis remains to be addressed. In this survey, we investigate the transcriptional dynamics of microRNAs and their target transcripts during neurogenesis of Drosophila melanogaster. To this end, we use the recently developed DIV-MARIS protocol, a method for enriching specific cell types from the Drosophila embryo in vivo, to sequence cell type-specific transcriptomes. We generate dedicated small and total RNA-seq libraries for neuroblasts, neurons and glia cells at early (6-8 h after egg laying (AEL)) and late (18-22 h AEL) stage. This allows us to directly compare these transcriptomes and investigate the potential functional roles of individual microRNAs with spatiotemporal resolution genome-wide, which is beyond the capabilities of existing in situ hybridization methods. Overall, we identify 74 microRNAs that are significantly differentially expressed between the three cell types and the two developmental stages. In all cell types, predicted target genes of down-regulated microRNAs show a significant enrichment of Gene Ontology terms related to neurogenesis. We also investigate how microRNAs regulate the transcriptome by targeting transcription factors and find many candidate microRNAs with putative roles in neurogenesis. Our survey highlights the roles of microRNAs as regulators of differentiation and glioneurognesis in the fruit fly and provides distinct starting points for dedicated functional follow-up studies.

Ballistic and Collisional Flow Contributions to Anti-Fourier Heat Transfer in Rarefied Cavity Flow.

This paper investigates anti-Fourier heat transfer phenomenon in a rarefied gas confined within a lid-driven cavity using a novel flow decomposition technique in the direct simulation Monte Carlo (DSMC) method proposed by Stefanov and co-workers. An isothermal cavity with different degrees of flow rarefaction from near continuum to mid transition regimes was considered to investigate cold-to-hot heat transfer from ballistic/collision flow decomposition viewpoint. A new cold-to-hot heat transfer indicator in the form of a scalar product of normalized heat flow vector and normalized temperature gradient vector has been introduced for the overall, ballistic and collision parts of these vectors. Using the new indicator, contributions of ballistic and collision flow parts to temperature and heat flux components was investigated with a specific emphasis on the cold-to-hot heat transfer phenomenon. We demonstrated that both ballistic and collision flow parts contribute to the occurrence of cold-to-hot heat transfer. However, it was found out that considered separately both ballistic and collision parts of heat transfer, when related to corresponding ballistic and collision temperature fields, they are ever hot-to-cold for all degrees of flow rarefaction. Thus, cold-to-hot heat transfer is a result of a subtle interplay between ballistic and collision parts in the slip and transition Knudsen regimes.

The Bulgarian version of the Juvenile Arthritis Multidimensional Assessment Report (JAMAR).

The Juvenile Arthritis Multidimensional Assessment Report (JAMAR) is a new parent/patient reported outcome measure that enables a thorough assessment of the disease status in children with juvenile idiopathic arthritis (JIA). We report the results of the cross-cultural adaptation and validation of the parent and patient versions of the JAMAR in the Bulgarian language. The reading comprehension of the questionnaire was tested in 10 JIA parents and patients. Each participating centre was asked to collect demographic, clinical data, and the JAMAR in 100 consecutive JIA patients or all consecutive patients seen in a 6-month period and to administer the JAMAR to 100 healthy children and their parents. The statistical validation phase explored descriptive statistics and the psychometric issues of the JAMAR: the 3 Likert assumptions, floor/ceiling effects, internal consistency, Cronbach's alpha, interscale correlations, test-retest reliability, and construct validity (convergent and discriminant validity). A total of 183 JIA patients (12% systemic, 53.6% oligoarticular, 23.5% RF negative polyarthritis, 10.9% other categories) and 100 healthy children were enrolled in two centres. The JAMAR components discriminated well healthy subjects from JIA patients. Notably, there is no significant difference between the healthy subjects and their affected peers in the school-related problems variable. All JAMAR components revealed good psychometric performances. In conclusion, the Bulgarian version of the JAMAR is a valid tool for the assessment of children with JIA and is suitable for use both in routine clinical practice and clinical research.

Growth and characterization of SiGeSn quantum well photodiodes.

We report on the fabrication and electro-optical characterization of SiGeSn multi-quantum well PIN diodes. Two types of PIN diodes, in which two and four quantum wells with well and barrier thicknesses of 10 nm each are sandwiched between B- and Sb-doped Ge-regions, were fabricated as single-mesa devices, using a low-temperature fabrication process. We discuss measurements of the diode characteristics, optical responsivity and room-temperature electroluminescence and compare with theoretical predictions from band structure calculations.

Predicting the Knudsen paradox in long capillaries by decomposing the flow into ballistic and collision parts.

The well-known Knudsen paradox observed in pressure driven rarefied gas flows through long capillaries is quantitatively explored by decomposing the particle distribution function into its ballistic and collision parts. The classical channel, tube, and duct Poiseuille flows are considered. The solution is obtained by a typical direct simulation Monte Carlo algorithm supplemented by a suitable particle decomposition indexation process. It is computationally confirmed that in the free-molecular and early transition regimes the reduction rate of the ballistic flow is larger than the increase rate of the collision flow deducing the Knudsen minimum of the overall flow. This description interprets in a precise, quantitative manner the appearance of the Knudsen minimum and verifies previously reported qualitative physical arguments.

Endovascular treatment of posttraumatic pseudoaneurysm of the common carotid artery.

Carotid artery injuries with pseudoaneurysm are uncommon but associated with central neurologic dysfunction. We present a case of posttraumatic pseudoaneurysm of the right common carotid artery treated by implantation of a covered stent. A 44-year-old woman with multiple injuries after fall from height presents a small dissection flap of the right common carotid artery (RCCA) on the initial computed tomography angiography (CTA). Fifteen days later a 10 mm pseudoaneurysm is observed on control CTA. We decided endovascular treatment. Through right femoral access with a long introducer sheath placed in the innominate artery, we implanted a covered stent Advanta V12 9 × 38 mm in the RCCA. The patient was discharged from the hospital with antiplatelet therapy without any neurological dysfunction and complete exclusion of the pseudoaneurysm. Use of covered stents has emerged as a safe and effective alternative to surgical repair of carotid injuries.

Thermal and second-law analysis of a micro- or nanocavity using direct-simulation Monte Carlo.

In this study the direct-simulation Monte Carlo (DSMC) method is utilized to investigate thermal characteristics of micro- or nanocavity flow. The rarefied cavity flow shows unconventional behaviors which cannot be predicted by the Fourier law, the constitutive relation for the continuum heat transfer. Our analysis in this study confirms some recent observations and shows that the gaseous flow near the top-left corner of the cavity is in a strong nonequilibrium state even within the early slip regime, Kn=0.005. As we obtained slip velocity and temperature jump on the driven lid of the cavity, we reported meaningful discrepancies between the direct and macroscopic sampling of rarefied flow properties in the DSMC method due to existence of nonequilibrium effects in the corners of cavity. The existence of unconventional nonequilibrium heat transfer mechanisms in the middle of slip regime, Kn=0.05, results in the appearance of cold-to-hot heat transfer in the microcavity. In the current study we demonstrate that existence of such unconventional heat transfer is strongly dependent on the Reynolds number and it vanishes in the large values of the lid velocity. As we compared DSMC solution with the results of regularized 13 moments (R13) equations, we showed that the thermal characteristic of the microcavity obtained by the R13 method coincides with the DSMC prediction. Our investigation also includes the analysis of molecular entropy in the microcavity to explain the heat transfer mechanism with the aid of the second law of thermodynamics. To this aim, we obtained the two-dimensional velocity distribution functions to report the molecular-based entropy distribution, and show that the cold-to-hot heat transfer in the cavity is well in accordance with the second law of thermodynamics and takes place in the direction of increasing entropy. At the end we introduce the entropy density for the rarefied flow and show that it can accurately illustrate departure from the equilibrium state.