The multi-dimensional challenges of controlling respiratory virus transmission in indoor spaces: Insights from the linkage of a microscopic pedestrian simulation and SARS-CoV-2 transmission model.

SARS-CoV-2 transmission in indoor spaces, where most infection events occur, depends on the types and duration of human interactions, among others. Understanding how these human behaviours interface with virus characteristics to drive pathogen transmission and dictate the outcomes of non-pharmaceutical interventions is important for the informed and safe use of indoor spaces. To better understand these complex interactions, we developed the Pedestrian Dynamics-Virus Spread model (PeDViS), an individual-based model that combines pedestrian behaviour models with virus spread models incorporating direct and indirect transmission routes. We explored the relationships between virus exposure and the duration, distance, respiratory behaviour, and environment in which interactions between infected and uninfected individuals took place and compared this to benchmark 'at risk' interactions (1.5 metres for 15 minutes). When considering aerosol transmission, individuals adhering to distancing measures may be at risk due to the buildup of airborne virus in the environment when infected individuals spend prolonged time indoors. In our restaurant case, guests seated at tables near infected individuals were at limited risk of infection but could, particularly in poorly ventilated places, experience risks that surpass that of benchmark interactions. Combining interventions that target different transmission routes can aid in accumulating impact, for instance by combining ventilation with face masks. The impact of such combined interventions depends on the relative importance of transmission routes, which is hard to disentangle and highly context dependent. This uncertainty should be considered when assessing transmission risks upon different types of human interactions in indoor spaces. We illustrated the multi-dimensionality of indoor SARS-CoV-2 transmission that emerges from the interplay of human behaviour and the spread of respiratory viruses. A modelling strategy that incorporates this in risk assessments can help inform policy makers and citizens on the safe use of indoor spaces with varying inter-human interactions.

IAnimal: a cross-species omics knowledgebase for animals.

With the exponential growth of multi-omics data, its integration and utilization have brought unprecedented opportunities for the interpretation of gene regulation mechanisms and the comprehensive analyses of biological systems. IAnimal (https://ianimal.pro/), a cross-species, multi-omics knowledgebase, was developed to improve the utilization of massive public data and simplify the integration of multi-omics information to mine the genetic mechanisms of objective traits. Currently, IAnimal provides 61 191 individual omics data of genome (WGS), transcriptome (RNA-Seq), epigenome (ChIP-Seq, ATAC-Seq) and genome annotation information for 21 species, such as mice, pigs, cattle, chickens, and macaques. The scale of its total clean data has reached 846.46 TB. To better understand the biological significance of omics information, a deep learning model for IAnimal was built based on BioBERT and AutoNER to mine 'gene' and 'trait' entities from 2 794 237 abstracts, which has practical significance for comprehending how each omics layer regulates genes to affect traits. By means of user-friendly web interfaces, flexible data application programming interfaces, and abundant functional modules, IAnimal enables users to easily query, mine, and visualize characteristics in various omics, and to infer how genes play biological roles under the influence of various omics layers.

DNMT1-targeting remodeling global DNA hypomethylation for enhanced tumor suppression and circumvented toxicity in oral squamous cell carcinoma.

BACKGROUND: The faithful maintenance of DNA methylation homeostasis indispensably requires DNA methyltransferase 1 (DNMT1) in cancer progression. We previously identified DNMT1 as a potential candidate target for oral squamous cell carcinoma (OSCC). However, how the DNMT1- associated global DNA methylation is exploited to regulate OSCC remains unclear. METHODS: The shRNA-specific DNMT1 knockdown was employed to target DNMT1 on oral cancer cells in vitro, as was the use of DNMT1 inhibitors. A xenografted OSCC mouse model was established to determine the effect on tumor suppression. High-throughput microarrays of DNA methylation, bulk and single-cell RNA sequencing analysis, multiplex immunohistochemistry, functional sphere formation and protein immunoblotting were utilized to explore the molecular mechanism involved. Analysis of human samples revealed associations between DNMT1 expression, global DNA methylation and collaborative molecular signaling with oral malignant transformation. RESULTS: We investigated DNMT1 expression boosted steadily during oral malignant transformation in human samples, and its inhibition considerably minimized the tumorigenicity in vitro and in a xenografted OSCC model. DNMT1 overexpression was accompanied by the accumulation of cancer-specific DNA hypomethylation during oral carcinogenesis; conversely, DNMT1 knockdown caused atypically extensive genome-wide DNA hypomethylation in cancer cells and xenografted tumors. This novel DNMT1-remodeled DNA hypomethylation pattern hampered the dual activation of PI3K-AKT and CDK2-Rb and inactivated GSK3ß collaboratively. When treating OSCC mice, targeting DNMT1 achieved greater anticancer efficacy than the PI3K inhibitor, and reduced the toxicity of blood glucose changes caused by the PI3K inhibitor or combination of PI3K and CDK inhibitors as well as adverse insulin feedback. CONCLUSIONS: Targeting DNMT1 remodels a novel global DNA hypomethylation pattern to facilitate anticancer efficacy and minimize potential toxic effects via balanced signaling synergia. Our study suggests DNMT1 is a crucial gatekeeper regarding OSCC destiny and treatment outcome.

Ionizing radiation-induced "zombie" carcinoma-associated fibroblasts with suppressed pro-radioresistance on OSCC cells.

OBJECTIVES: This study was to investigate the effect of ionizing radiation (IR) on oral carcinoma-associated fibroblasts (CAFs) and to further explore subsequent effects of IR-induced "zombie" CAFs on oral squamous cell carcinoma (OSCC) cells. MATERIALS AND METHODS: Three primary CAFs and one primary normal-associated fibroblasts (NAFs) were separated from human OSCC and normal oral mucosa tissues, identified by immunocytochemistry. Cells were exposed to IR by X-ray irradiator under different doses. The DNA damage, proliferation, and migration of irradiated CAFs were, respectively, detected by immunofluorescence and wound healing assay, while senescence was detected by ß-galactosidase staining. Finally, the effect of irradiated CAFs on biological behavior and radioresistance of Cal-27 cells were determined via assays mentioned above. RESULTS: Oral CAFs were sensitive to IR with DNA damage increasing and proliferation decreasing. 18 Gy IR could not kill oral CAFs but induce them to "zombies," with arrested proliferation, increased senescence, and reduced migration. "Zombie" CAFs (zCAFs) could enhance proliferation, migration, and invasion of Cal-27 cells, and show suppressed pro-radioresistance by reducing DNA damage and facilitating survival. CONCLUSIONS: IR-induced zCAFs could continuously promote radioresistance of OSCC cells though being suppressed, suggesting the potential promoting effect on tumor relapse post-radiotherapy that needed further exploring.

Significant efficacy of short-course, low-concentration betamethasone mouthwash therapy for severe erosive oral lichen planus: a randomized controlled trial.

OBJECTIVES: To evaluate the short-term efficacy of low-concentration betamethasone mouthwash for severe erosive oral lichen planus (EOLP). MATERIALS AND METHOD: In this randomized, investigator-blind, positive-controlled trial, OLP patients with erosive lesions received betamethasone mouthwash (0.137 mg/mL) or dexamethasone mouthwash (0.181 mg/mL) three times daily for 2 or 4 weeks and were followed up for 3 months to observe recurrence. The primary outcome was the week-2 reduction in erosive area. RESULTS: Fifty-seven participants were randomized to betamethasone (n = 29) and dexamethasone (n = 28). At week 2, participants using betamethasone (n = 28) experienced a greater reduction in erosive area than gargling with dexamethasone (n = 26). Similarly, secondary outcomes, including the healing proportion of erosions, reduced pain level, reduction in atrophic area, Thongprasom score, and recurrence interval, showed the superiority of betamethasone. At week 4, betamethasone (n = 7) was not superior to dexamethasone (n = 15) in further reducing lesional area and pain level. No serious adverse events were documented. CONCLUSIONS: The 0.137 mg/mL compound betamethasone mouthwash exhibited significant efficacy in rapidly enhancing erosion healing within 2 weeks and extending the recurrence interval with a good safety profile. CLINICAL RELEVANCE: This study proved the significant efficacy of short-course 0.137 mg/mL betamethasone mouthwash therapy for treating erosion and pain, providing a novel topical agent for patients with severe EOLP. TRIAL REGISTRATION: This study was prospectively registered at the International Clinical Trials Registry Platform ( ChiCTR1800016507 ) on 5 June 2018.

Multidisciplinary approaches for elucidating genetics and molecular pathogenesis of urinary tract malformations.

Advances in clinical diagnostics and molecular tools have improved our understanding of the genetically heterogeneous causes underlying congenital anomalies of kidney and urinary tract (CAKUT). However, despite a sharp incline of CAKUT reports in the literature within the past 2 decades, there remains a plateau in the genetic diagnostic yield that is disproportionate to the accelerated ability to generate robust genome-wide data. Explanations for this observation include (i) diverse inheritance patterns with incomplete penetrance and variable expressivity, (ii) rarity of single-gene drivers such that large sample sizes are required to meet the burden of proof, and (iii) multigene interactions that might produce either intra- (e.g., copy number variants) or inter- (e.g., effects in trans) locus effects. These challenges present an opportunity for the community to implement innovative genetic and molecular avenues to explain the missing heritability and to better elucidate the mechanisms that underscore CAKUT. Here, we review recent multidisciplinary approaches at the intersection of genetics, genomics, in vivo modeling, and in vitro systems toward refining a blueprint for overcoming the diagnostic hurdles that are pervasive in urinary tract malformation cohorts. These approaches will not only benefit clinical management by reducing age at molecular diagnosis and prompting early evaluation for comorbid features but will also serve as a springboard for therapeutic development.

Unraveling the Electronic Effect of Transition-Metal Dopants (M = Fe, Co, Ni, and Cu) and Graphene Substrate on Platinum-Transition Metal Dimers for Hydrogen Evolution Reaction.

As an extension of single-atom catalysts, despite the increased opportunities to optimize the hydrogen evolution reaction (HER) activity with the variation of the composition, dual-metal-atom catalysts, i.e., dimers, are deeply trapped in a design blind spot due to the lack of the essential recognition of the intrinsic catalytic mechanism at the atomic level. Herein, based on first-principles calculations, a series of platinum-transition metal dimers were constructed on nitrogen-doped graphene (PtM-NDG, M = Fe, Co, Ni, Cu) to reveal the effects of the internal (i.e., M atom) and external (i.e., NDG substrate) environments on the HER activity. Computational results show that the original over-adsorption of hydrogen intermediate (H*) of PtM dimer is weakened after the introduction of NDG, and the optimal active site migrates from the Pt in PtM dimer to the Pt-M bridge in PtM-NDG, triggered by the redistribution of the charge density of the metal atoms. In particular, the M atom switches from tuning the d-band center of the Pt atom to indirectly assist the adsorption behavior of Pt in the PtM dimer to the direct participation in the bonding with H* in PtM-NDG via its own d-band to regulate the distribution of σ and σ*, which enables fine modulation of the bond strength with H*. Moreover, the overall hydrogen evolution performance of PtM-NDG is mainly determined by the d-band center of the M atom. Furthermore, PtFe-NDG with the lowest energy barrier of the rate-determining step stands out in the process of H2 desorption and water dissociation. The present work deepens our understanding of the effects of the metal dopant and substrate on the catalytic performance of platinum.

Differential OAT methylation correlates with cell infiltration in tumor microenvironment and overall survival postradiotherapy in oral squamous cell carcinoma patient.

BACKGROUND: Given that DNA methylation and tumor microenvironment (TME) are susceptible to radiotherapy, we aimed to figure out specific differential DNA methylation to reflect oral squamous cell carcinoma (OSCC) prognosis and associated effect on TME changes postradiotherapy, performing as an efficient biomarker. MATERIALS AND METHODS: Differentially methylation analysis was performed using data from The Cancer Genome Atlas. Curves of Kaplan-Meier (K-M) survival, cumulative hazard and events, Cox proportional hazards, and linear regression model were conducted to screen and validate differential methylation genes, while multiple regression equation to analyze if ornithine aminotransferase (OAT) methylation correlates with radiotherapy. For correlation between OAT methylation and immune infiltrates, CIBERSORT and ESTIMATE algorithms were performed, following gene set enrichment analysis (GSEA) and ssGSEA analysis to evaluate biological process. RESULTS: Compared to normal tissues, only OAT in OSCC was differential significantly by K-M analysis (p = 0.0364). OAT hypermethylation was associated with increased overall survival (hazard ratio: 0.65, p = 0.0358). Radiotherapy correlated with OAT methylation (ß = -0.01, p = 0.0061); most patients with OAT hypermethylation were radiation-sensitive. Hypomethylated OAT correlated with higher cell infiltrations in TME. Neuroactive ligand-receptor interaction was most significantly related to OAT methylation (p = 9.2e-10). Sulfur metabolism was the most significantly in OAT hypermethylation group (p = 0.0041) and RIG-I-like receptor in OAT hypomethylation group (p = 0.0094). CONCLUSION: OAT methylation can serve as a predictor of OSCC prognosis postradiotherapy with potential mechanism by changing cell infiltrations in TME, but further experimental study deserves to carry out confirming the role and mechanism of OAT methylation in OSCC.

A numerically stable constrained optimal filter design method for multichannel active noise control using dual conic formulation.

In practical active noise control (ANC) applications, various constraints are usually required, for example, the disturbance enhancement constraint, the robust stability constraint, and the controller output power constraint. One commonly used approach for designing a constrained ANC filter is to formulate a constrained optimization problem using an H / H framework, which requires significant computational power to solve. Recent work has shown that such an ANC filter design problem can be reformulated into a convex optimization problem and then further reformulated to a cone programming problem to reduce the required computational time by several orders. However, the standard cone programming reformulation procedure leads to a large number of free variables, which, in many applications, can adversely influence the numerical behavior of the optimization algorithm. In the current work, the ANC filter design problem structure is exploited in its dual conic form, which allows an elimination of free variables and can result in a numerically more stable solving process for the filter design problem while keeping the noise control performance unchanged. It is demonstrated that when compared with the reformulation using the standard procedure, the proposed formulation significantly improves its numerical stability and computational efficiency.

Loss of δ-catenin function in severe autism.

Autism is a multifactorial neurodevelopmental disorder affecting more males than females; consequently, under a multifactorial genetic hypothesis, females are affected only when they cross a higher biological threshold. We hypothesize that deleterious variants at conserved residues are enriched in severely affected patients arising from female-enriched multiplex families with severe disease, enhancing the detection of key autism genes in modest numbers of cases. Here we show the use of this strategy by identifying missense and dosage sequence variants in the gene encoding the adhesive junction-associated δ-catenin protein (CTNND2) in female-enriched multiplex families and demonstrating their loss-of-function effect by functional analyses in zebrafish embryos and cultured hippocampal neurons from wild-type and Ctnnd2 null mouse embryos. Finally, through gene expression and network analyses, we highlight a critical role for CTNND2 in neuronal development and an intimate connection to chromatin biology. Our data contribute to the understanding of the genetic architecture of autism and suggest that genetic analyses of phenotypic extremes, such as female-enriched multiplex families, are of innate value in multifactorial disorders.

Constrained optimal filter design for multi-channel active noise control via convex optimization.

In many practical multi-channel active noise control (ANC) applications, various constraints need to be satisfied, such as stability, enhancement, etc. One way to enforce these constraints is to add a regularization term to the Wiener filter formulation, which, by tuning only a single parameter, can over satisfy many constraints and degrade the ANC performance. Another approach for non-adaptive ANC filter design that can produce better ANC performance is to directly solve the constrained optimization problem formulated based on the H2/H∞ control framework. However, such a formulation does not result in a convex optimization problem and its practicality can be limited by the significant computation time required in the solving process. In the presented work, the H2/H∞ formulation is convexified and a global minimum is guaranteed. It is then further reformulated into a cone programming problem which can be solved using specialized algorithms. Results show that the proposed method can produce better noise control performance than the regularization method. Compared with the traditional H2/H∞ formulation, the proposed method is more reliable and the computation time can be reduced by several orders, which, practically, provides a potential to extend its application to adaptive control.

Genetic Drivers of Kidney Defects in the DiGeorge Syndrome.

BACKGROUND: The DiGeorge syndrome, the most common of the microdeletion syndromes, affects multiple organs, including the heart, the nervous system, and the kidney. It is caused by deletions on chromosome 22q11.2; the genetic driver of the kidney defects is unknown. METHODS: We conducted a genomewide search for structural variants in two cohorts: 2080 patients with congenital kidney and urinary tract anomalies and 22,094 controls. We performed exome and targeted resequencing in samples obtained from 586 additional patients with congenital kidney anomalies. We also carried out functional studies using zebrafish and mice. RESULTS: We identified heterozygous deletions of 22q11.2 in 1.1% of the patients with congenital kidney anomalies and in 0.01% of population controls (odds ratio, 81.5; P=4.5×10-14). We localized the main drivers of renal disease in the DiGeorge syndrome to a 370-kb region containing nine genes. In zebrafish embryos, an induced loss of function in snap29, aifm3, and crkl resulted in renal defects; the loss of crkl alone was sufficient to induce defects. Five of 586 patients with congenital urinary anomalies had newly identified, heterozygous protein-altering variants, including a premature termination codon, in CRKL. The inactivation of Crkl in the mouse model induced developmental defects similar to those observed in patients with congenital urinary anomalies. CONCLUSIONS: We identified a recurrent 370-kb deletion at the 22q11.2 locus as a driver of kidney defects in the DiGeorge syndrome and in sporadic congenital kidney and urinary tract anomalies. Of the nine genes at this locus, SNAP29, AIFM3, and CRKL appear to be critical to the phenotype, with haploinsufficiency of CRKL emerging as the main genetic driver. (Funded by the National Institutes of Health and others.).

Construction of Bridged Carbocycles and Heterocycles via Rh(III)-Catalyzed C-H Alkylation/Michael Addition of 2-Arylindoles with Quinone Monoacetals.

A rhodium-catalyzed formal [4 + 5] annulation reaction of 2-arylindoles with quinone monoacetals for the selective preparation of bridged nine-membered carbocyclic and heterocyclic compounds is developed. When 2-aryl substituted indoles are employed, this annulation reaction affords bridged nine-membered carbocyclic compounds with excellent indolyl C3 selectivity. On the other hand, with 2-aryl-3-substituted indoles as the substrates, bridged nine-membered heterocyclic compounds are exclusively formed via N1 annulation. Further transformations of the obtained products into new bridged compounds showcase the synthetic potential of this protocol.

Spatially sparse sound source localization in an under-determined system by using a hybrid compressive sensing method.

Near-field Acoustical Holography is a powerful tool for sound source identification and sound field reconstruction. Generally, many microphone measurements are required to construct a source model that can span the whole sound source region while simultaneously avoiding measurement errors and ensuring a high spatial sampling rate. That type of measurement is economically costly and hard to perform in industrial environments. Motivated by the desire to be able to use a relatively small number of microphone measurements to reconstruct a sound field and to accurately identify sound source locations, an Equivalent Source Method (ESM) is considered here. In particular, the focus is on the combination of a monopole-distribution ESM and a relatively small number of microphone measurements, thus creating an under-determined system. Wideband Acoustical Holography and l1-norm Convex Optimization are introduced to solve this under-determined inverse problem. Based on the attributes of these two methods, a hybrid method combining the best features of each is proposed to identify sound source locations.

Calculation of acoustic radiation modes by using spherical waves and generalized singular value decomposition.

Acoustic radiation modes (ARMs) have been widely used in noise control engineering owing to their convenient sound-power characteristics. However, the evaluation of ARMs for non-regular structures can be computationally intensive: it usually involves solving the boundary integral equation (BIE) by using the boundary element method in order to obtain the sound power radiation resistance matrix, followed by a singular value decomposition to obtain the radiation modes. The proposed procedure involves projecting spherical harmonics onto an enclosing surface, followed by the application of generalized singular value decomposition, with the result that the need to solve the BIE is eliminated, which potentially reduces the computational effort significantly.

Involvement of BIG5 and BIG3 in BRI1 Trafficking Reveals Diverse Functions of BIG-subfamily ARF-GEFs in Plant Growth and Gravitropism.

ADP-ribosylation factor-guanine nucleotide exchange factors (ARF-GEFs) act as key regulators of vesicle trafficking in all eukaryotes. In Arabidopsis, there are eight ARF-GEFs, including three members of the GBF1 subfamily and five members of the BIG subfamily. These ARF-GEFs have different subcellular localizations and regulate different trafficking pathways. Until now, the roles of these BIG-subfamily ARF-GEFs have not been fully revealed. Here, analysis of the BIGs expression patterns showed that BIG3 and BIG5 have similar expression patterns. big5-1 displayed a dwarf growth and big3-1 big5-1 double mutant showed more severe defects, indicating functional redundancy between BIG3 and BIG5. Moreover, both big5-1 and big3-1 big5-1 exhibited a reduced sensitivity to Brassinosteroid (BR) treatment. Brefeldin A (BFA)-induced BR receptor Brassinosteroid insensitive 1 (BRI1) aggregation was reduced in big5-1 mutant, indicating that the action of BIG5 is required for BRI1 recycling. Furthermore, BR-induced dephosphorylation of transcription factor BZR1 was decreased in big3-1 big5-1 double mutants. The introduction of the gain-of-function of BZR1 mutant BZR1-1D in big3-1 big5-1 mutants can partially rescue the big3-1 big5-1 growth defects. Our findings revealed that BIG5 functions redundantly with BIG3 in plant growth and gravitropism, and BIG5 participates in BR signal transduction pathway through regulating BRI1 trafficking.

A truncating mutation in CEP55 is the likely cause of MARCH, a novel syndrome affecting neuronal mitosis.

BACKGROUND: Hydranencephaly is a congenital anomaly leading to replacement of the cerebral hemispheres with a fluid-filled cyst. The goals of this work are to describe a novel autosomal-recessive syndrome that includes hydranencephaly (multinucleated neurons, anhydramnios, renal dysplasia, cerebellar hypoplasia and hydranencephaly (MARCH)); to identify its genetic cause(s) and to provide functional insight into pathomechanism. METHODS: We used homozygosity mapping and exome sequencing to identify recessive mutations in a single family with three affected fetuses. Immunohistochemistry, RT-PCR and imaging in cell lines, and zebrafish models, were used to explore the function of the gene and the effect of the mutation. RESULTS: We identified a homozygous nonsense mutation in CEP55 segregating with MARCH. Testing the effect of this allele on patient-derived cells indicated both a reduction of the overall CEP55 message and the production of a message that likely gives rise to a truncated protein. Suppression or ablation of cep55l in zebrafish embryos recapitulated key features of MARCH, most notably renal dysplasia, cerebellar hypoplasia and craniofacial abnormalities. These phenotypes could be rescued by full-length but not truncated human CEP55 message. Finally, we expressed the truncated form of CEP55 in human cells, where we observed a failure of truncated protein to localise to the midbody, leading to abscission failure and multinucleated daughter cells. CONCLUSIONS: CEP55 loss of function mutations likely underlie MARCH, a novel multiple congenital anomaly syndrome. This association expands the involvement of centrosomal proteins in human genetic disorders by highlighting a role in midbody function.

Sound field reconstruction using multipole equivalent source model with un-fixed source locations.

The equivalent source methods (ESM) that have been developed to this point can generally be classified into two categories: one in which a relatively large number of lower order sources are fixed at various locations, and one in which a series of higher order sources are fixed at one location. The present work started with a model in the latter category, but the individual sources were then allowed to move separately to locations which were determined by using a nonlinear optimization procedure based on the measured sound field data. To test this approach, experiments were conducted using a small loudspeaker cabinet; measurements were made using an array of microphones on all sides of the loudspeaker. It was found that by allowing the source components to move, the sound field representation in both the near and far fields was improved, particularly at high frequencies, compared to the model with fixed source locations. By comparison with results obtained from boundary element calculations based on laser vibrometer measurements of the loudspeaker's diaphragm and tweeter velocities, it was found that the proposed ESM can also be used to accurately predict the sound pressure distribution on the source surface.

A novel test for recessive contributions to complex diseases implicates Bardet-Biedl syndrome gene BBS10 in idiopathic type 2 diabetes and obesity.

Rare-variant association studies in common, complex diseases are customarily conducted under an additive risk model in both single-variant and burden testing. Here, we describe a method to improve detection of rare recessive variants in complex diseases termed RAFT (recessive-allele-frequency-based test). We found that RAFT outperforms existing approaches when the variant influences disease risk in a recessive manner on simulated data. We then applied our method to 1,791 Finnish individuals with type 2 diabetes (T2D) and 2,657 matched control subjects. In BBS10, we discovered a rare variant (c.1189A>G [p.Ile397Val]; rs202042386) that confers risk of T2D in a recessive state (p = 1.38 × 10(-6)) and would be missed by conventional methods. Testing of this variant in an established in vivo zebrafish model confirmed the variant to be pathogenic. Taken together, these data suggest that RAFT can effectively reveal rare recessive contributions to complex diseases overlooked by conventional association tests.