Targeted Repair of Spinal Cord Injury Based on miRNA-124-3p-Loaded Mesoporous Silica Camouflaged by Stem Cell Membrane Modified with Rabies Virus Glycoprotein.

Spinal cord injury (SCI) has no effective treatment modalities. It faces a significant global therapeutical challenge, given its features of poor axon regeneration, progressive local inflammation, and inefficient systemic drug delivery due to the blood-spinal cord barrier (BSCB). To address these challenges, a new nano complex that achieves targeted drug delivery to the damaged spinal cord is proposed, which contains a mesoporous silica nanoparticle core loaded with microRNA and a cloaking layer of human umbilical cord mesenchymal stem cell membrane modified with rabies virus glycoprotein (RVG). The nano complex more readily crosses the damaged BSCB with its exosome-resembling properties, including appropriate size and a low-immunogenic cell membrane disguise and accumulates in the injury center because of RVG, where it releases abundant microRNAs to elicit axon sprouting and rehabilitate the inflammatory microenvironment. Culturing with nano complexes promotes axonal growth in neurons and M2 polarization in microglia. Furthermore, it showed that SCI mice treated with this nano complex by tail vein injection display significant improvement in axon regrowth, microenvironment regulation, and functional restoration. The efficacy and biocompatibility of the targeted delivery of microRNA by nano complexes demonstrate their immense potential as a noninvasive treatment for SCI.

Magnetic Nanoparticles and Methylprednisolone Based Physico-Chemical Bifunctional Neural Stem Cells Delivery System for Spinal Cord Injury Repair.

Neural stem cells (NSCs) transplantation is an attractive and promising treatment strategy for spinal cord injury (SCI). Various pathological processes including the severe inflammatory cascade and difficulty in stable proliferation and differentiation of NSCs limit its application and translation. Here, a novel physico-chemical bifunctional neural stem cells delivery system containing magnetic nanoparticles (MNPs and methylprednisolone (MP) is designed to repair SCI, the former regulates NSCs differentiation through magnetic mechanical stimulation in the chronic phase, while the latter alleviates inflammatory response in the acute phase. The delivery system releases MP to promote microglial M2 polarization, inhibit M1 polarization, and reduce neuronal apoptosis. Meanwhile, NSCs tend to differentiate into functional neurons with magnetic mechanical stimulation generated by MNPs in the static magnetic field, which is related to the activation of the PI3K/AKT/mTOR pathway. SCI mice achieve better functional recovery after receiving NSCs transplantation via physico-chemical bifunctional delivery system, which has milder inflammation, higher number of M2 microglia, more functional neurons, and axonal regeneration. Together, this bifunctional NSCs delivery system combined physical mechanical stimulation and chemical drug therapy is demonstrated to be effective, which provides new treatment insights into clinical transformation of SCI repair.

Multifunctional Conductive and Electrogenic Hydrogel Repaired Spinal Cord Injury via Immunoregulation and Enhancement of Neuronal Differentiation.

Spinal cord injury (SCI) is a refractory neurological disorder. Due to the complex pathological processes, especially the secondary inflammatory cascade and the lack of intrinsic regenerative capacity, it is difficult to recover neurological function after SCI. Meanwhile, simulating the conductive microenvironment of the spinal cord reconstructs electrical neural signal transmission interrupted by SCI and facilitates neural repair. Therefore, a double-crosslinked conductive hydrogel (BP@Hydrogel) containing black phosphorus nanoplates (BP) is synthesized. When placed in a rotating magnetic field (RMF), the BP@Hydrogel can generate stable electrical signals and exhibit electrogenic characteristic. In vitro, the BP@Hydrogel shows satisfactory biocompatibility and can alleviate the activation of microglia. When placed in the RMF, it enhances the anti-inflammatory effects. Meanwhile, wireless electrical stimulation promotes the differentiation of neural stem cells (NSCs) into neurons, which is associated with the activation of the PI3K/AKT pathway. In vivo, the BP@Hydrogel is injectable and can elicit behavioral and electrophysiological recovery in complete transected SCI mice by alleviating the inflammation and facilitating endogenous NSCs to form functional neurons and synapses under the RMF. The present research develops a multifunctional conductive and electrogenic hydrogel for SCI repair by targeting multiple mechanisms including immunoregulation and enhancement of neuronal differentiation.

Aggregation-Induced Emission Photosensitizer-Engineered Anticancer Nanomedicine for Synergistic Chemo/Chemodynamic/Photodynamic Therapy.

Photodynamic therapy (PDT) with aggregation-induced emission (AIE) photosensitizers (PSs) is a promising therapeutic strategy to achieve better anticancer results. However, eradicating solid tumors completely by PDT alone can be difficult owing to the inherent drawbacks of this treatment, and the combination of PDT with other therapeutic modalities provides opportunities to achieve cooperative enhancement interactions among various treatments. Herein, this work presents the construction of a biocompatible nanocomposite, namely CaO2@DOX@ZIF@ASQ, featuring light-responsive reactive oxygen species (ROS) generation and tumor-targeting oxygen and hydrogen peroxide discharge, as well as controlled doxorubicin (DOX) and copper ion release, thus allowing the combined PDT/CT/CDT effect by AIE PS-enhanced PDT, DOX-based chemotherapy (CT), and copper-involved Fenton-like reaction-driven chemodynamic therapy (CDT). In vitro and in vivo studies verify that the generation of both ROS and O2 by this nanomedicine, stimulated by light, exhibits superior anticancer efficacy, alleviating tumor hypoxia and achieving synergistic PDT/CT/CDT therapeutic effect. This multifunctional nanomedicine remarkably suppresses the tumor growth with minimized systemic toxicity, providing a new strategy for constructing multimodal PDT/CT/CDT therapeutic systems to overcome hypoxia limitations, and potentially increase the antitumor efficacy at lower doses of PSs and chemotherapeutic drugs, thus minimizing potential toxicity to non-malignant tissues.

Dynamic assessment of eco-civilization in Guangdong Province based on geographic information system (GIS) and analytic hierarchy process (AHP).

Based on the data from 2017 to 2021, this paper uses the organic combination of Geographic Information System (GIS) and Analytic Hierarchy Process (AHP) to conduct a dynamic evaluation of the standard of Guangdong's ecological civilization. The results clearly reveal that: (1) the Guangdong Province's ecological civilization is 46.31 % above the Chinese average, but growth is uneven across cities; (2) the fluctuation of the level of eco-civilization building in the province declines more obviously; (3) cities perform differently in different dimensions of eco-civilization building, most cities in Guangdong exhibited positive performance in terms of ecology and environment, but poor performance in terms of economic development and social construction; (4) economic benefits have a strong positive influence on the level of eco-civilization building in Guangdong Province. Finally, targeted suggestions are made for the construction of eco-civilization in Guangdong Province.

Machine learning-identified stemness features and constructed stemness-related subtype with prognosis, chemotherapy, and immunotherapy responses for non-small cell lung cancer patients.

AIM: This study aimed to explore a novel subtype classification method based on the stemness characteristics of patients with non-small cell lung cancer (NSCLC). METHODS: Based on the Cancer Genome Atlas database to calculate the stemness index (mRNAsi) of NSCLC patients, an unsupervised consensus clustering method was used to classify patients into two subtypes and analyze the survival differences, somatic mutational load, copy number variation, and immune characteristics differences between them. Subsequently, four machine learning methods were used to construct and validate a stemness subtype classification model, and cell function experiments were performed to verify the effect of the signature gene ARTN on NSCLC. RESULTS: Patients with Stemness Subtype I had better PFS and a higher somatic mutational burden and copy number alteration than patients with Stemness Subtype II. In addition, the two stemness subtypes have different patterns of tumor immune microenvironment. The immune score and stromal score and overall score of Stemness Subtype II were higher than those of Stemness Subtype I, suggesting a relatively small benefit to immune checkpoints. Four machine learning methods constructed and validated classification model for stemness subtypes and obtained multiple logistic regression equations for 22 characteristic genes. The results of cell function experiments showed that ARTN can promote the proliferation, invasion, and migration of NSCLC and is closely related to cancer stem cell properties. CONCLUSION: This new classification method based on stemness characteristics can effectively distinguish patients' characteristics and thus provide possible directions for the selection and optimization of clinical treatment plans.

Effect of pleural invasion on survival of patients with small cell lung cancer: Propensity score analysis and nomogram establishment based on the SEER database.

Objectives: Pleural invasion (PI) is identified as an adverse prognostic factor for non-small cell lung cancer (NSCLC), but its value in small cell lung cancer (SCLC) remains unclear. We aimed to evaluate the survival effect of PI on overall survival (OS) in SCLC, meanwhile, we established a predictive nomogram based on related risk factors for OS in SCLC patients with PI. Methods: We extracted the data of patients diagnosed with primary SCLC between 2010 and 2018 from the Surveillance, Epidemiology, and End Results (SEER) database. The propensity score matching (PSM) method was used to minimize the baseline difference between the non-PI and PI groups. Kaplan-Meier curves and the log-rank test were used for survival analysis. Univariate and multivariate Cox regression analyses were applied to identify the independent prognostic factors. Randomly divided the patients with PI into training (70%) and validation (30%) cohorts. A prognostic nomogram was established based on the training cohort and was evaluated in the validation cohort. The C-index, receiver operating characteristic curves (ROC), calibration curves, and decision curve analysis (DCA) were applied to assess the performance of the nomogram. Results: A total of 1,770 primary SCLC patients were enrolled, including1321patients with non-PI and 449 patients with PI. After PSM, the 387 patients in the PI group matched the 387 patients in the non-PI group. By Kaplan-Meier survival analysis, we observed the exact beneficial effect of non-PI on OS in both original and matched cohorts. Multivariate Cox analysis showed similar results to demonstrate a statistically significant benefit for patients with non-PI in both original and matched cohorts. Age, N stage, M stage, surgery, radiotherapy, and chemotherapy were independent prognostic factors for SCLC patients with PI. The C-index of the nomogram in the training and validation cohort was 0.714 and 0.746, respectively. The ROC curves, calibration curves, and DCA curves also demonstrated good predictive performance in the training and validation cohorts of the prognostic nomogram. Conclusion: Our study shows that PI is an independent poor prognostic factor for SCLC patients. The nomogram is a useful and reliable tool to predict the OS in SCLC patients with PI. The nomogram can provide strong references to clinicians to facilitate clinic decisions.

Caries prevalence of the first permanent molar and associated factors among second-grade students in Xiangyun of Yunnan, China: A cross-sectional study.

Purpose: This study aimed to explore the caries prevalence of the first permanent molar (FPM) and the associated factors among second-grade students in Xiangyun of Yunnan, China. Materials and methods: A cross-sectional survey was conducted in Xiangcheng Town, Xiangyun County, China, from September to November 2020. The methodology recommended by WHO was used for the dental examination. All the parents were required to complete a questionnaire to collect information on children's family background, dietary habits, oral health behaviors and parental oral health-related knowledge. The tests of significance used were the chi-square test. The association between dental caries prevalence and its associated factors was investigated using logistic regression analysis. Statistical significance was set at P < 0.05. Results: Data of 1,295 second graders consisting of 665 males and 630 females were analyzed. The caries prevalence of FPM was 47.6%, and the mean DMFT was 1.11 ± 1.394 in this sample. Among all the children with caries, the filling rate is meager, 2.6%. There were statistically significant differences in the caries prevalence of FPM among age groups. No significant difference existed between the sexes. Logistic regression analysis showed that the most significantly associated factors were: consuming desserts at least once a day, no fluoride varnish application experience, worse parental perception of children's oral health status, and incorrect parental knowledge of whether decayed primary teeth need treatment. Conclusion: Caries prevalence of FPM among second-grade schoolchildren in Xiangyun was considerably higher than the average prevalence nationwide in China. As China aims to reduce dental caries to <25% of 12-year-old children in 2030, the local government of remote regions like Xiangyun needs to do much more to achieve this goal. Results of this study could provide a framework for ongoing and future public oral health programs and policy planning in Xiangyun, with particular attention to early preventive and therapeutic measures.

Early childhood caries prevalence and associated factors among preschoolers aged 3-5 years in Xiangyun, China: A cross-sectional study.

Purpose: This study aimed to investigate the early childhood caries (ECC) prevalence among preschoolers aged 3-5 years in Xiangyun of Yunnan, China and explore the factors associated with the prevalence. Materials and methods: A cross-sectional survey including 3-5-year-old children was conducted in Xiangyun County, China, between September and November 2020. According to the dental examination standard of the WHO 2013 criteria, the presence of ECC was recorded. Besides the dental examination for children, their parents completed questionnaires about caries-related factors, including demographic variables, family socioeconomic status, feeding, and oral health-related habits, parental dental knowledge, and the condition of dental service utilization. SPSS Statistics 25.0 (IBM, Chicago, IL, USA) was used for data analysis. Statistical significance was set at p < 0.05. Results: The ECC prevalence among a sample of 1,280 children aged 3-5 years consisting of 665 boys and 615 girls in this study, was 74.3%, and the mean decayed-missing-filled teeth (dmft) was 4.9 ± 5.0. There were no statistically significant differences in the ECC prevalence between the sexes and among different ethnic groups. Children with different dietary and oral hygiene habits showed no significantly different prevalence of ECC. Logistic regression analysis showed that the most significantly associated factors were older age, lower family income, and worse parental perception of children's oral health status. Conclusion: The ECC prevalence among 3-5-year-old preschool children in Xiangyun was higher than the average national ECC prevalence in China. This study implies that more attention should be given to children's caries prevention from early childhood; oral health education and promotion should be intensified to reduce the ECC prevalence and improve the oral health status of children in Xiangyun.

Characteristics of Oral Mucosal Lesions and Their Association With Socioeconomic Status and Systemic Health: A Cross-Sectional Study of Consecutively Collected Oral Medicine Clinic Data in a Remote Rural Area of China.

Objectives: Epidemiological data of oral mucosal lesions (OMLs) are required to develop practical oral care policies. However, limited data are available for rural areas in China. We aimed to estimate the spectrum and frequency of OMLs and to identify their associated socioeconomic status (SES) and systemic health in a remote rural area in Yunnan, China. Methods: We screened patients for OMLs in an oral medicine clinic in rural Yunnan, China, from August 2020 to February 2021. OMLs were documented. SES, including the highest education level achieved and the previous month's household income, as well as the patient's systemic health, including a history of smoking, alcohol use, and chronic disease, were obtained from the Medical History/Health Questionnaire Form and patient medical records. Results: A total of 146 patients were found to have OMLs. The most frequent OML was aphthous ulcer (n = 41, 28.1%), followed by burning mouth syndrome (BMS) (n = 16, 11.0%), viral ulcer (n = 14, 9.6%), and oral lichen planus (OLP) (n = 9, 6.2%). In these patients, the most common chronic diseases were sleep apnea (n = 35, 24.0%), hypertension (n = 23, 15.8%), bronchitis (n = 16, 11.0%), reflux (including gastroesophageal reflux disease) (n = 12, 8.2%), and arthritis (n = 11, 7.5%). On adjusted regression, the patients without chronic diseases had a lower risk of BMS than those with chronic diseases [odds ratio (OR), 0.2; 95% confidence interval (CI), 0.03-0.9]. Age was an independent protective factor for viral ulcers (OR, 1.0; 95% CI, 0.9-1.0). Patients with low-income levels had a lower risk of OLP than those with high-income levels (OR, 0.2; 95% CI, 0.05-0.9). Conclusions: Our oral medicine clinic data in remote Yunnan, China, showed the most frequent OML was aphthous ulcer, which was followed by BMS, viral ulcer, and OLP. Oral care policies should be prioritized among patients with aphthous ulcer. Preventive strategy of BMS should be targeted to people with chronic disease for health equalities. Therefore, an individualized strategy for interventions of OMLs might be optimal, based on the specific epidemiologic characteristics in rural settings.

Prevalence of oral mucosal lesions in children in Xiangyun of Yunnan, China: a cross-sectional study.

BACKGROUND: This population-based cross-sectional study aimed to evaluate the prevalence of oral mucosal lesions (OMLs) in children in a rural area in China as epidemiological data on these conditions from such areas are insufficient. METHODS: A total of 3145 children in Xiangyun of Yunnan were enrolled. A socio-economic questionnaire and a field survey of OMLs were administered. We gathered information on factors (sex, age, caregiver, parental education, and last-month household income) that might be associated with OMLs in these children. RESULTS: OMLs in children in Xiangyun of Yunnan had a prevalence of 1.8% (95% CI; 1.3-2.3%). The most prevalent OML was oral ulcer (n = 11; 18.3%), followed by linea alba (n = 10; 16.7%), whereas the least prevalent OMLs were frenal tag (n = 1; 1.7%) and herpes labialis (n = 1; 1.7%). On unadjusted or adjusted regression, school-aged children had about 50% lower risk of OMLs than preschoolers. CONCLUSIONS: These findings indicate that the prevalence of OMLs in Xiangyun of Yunnan is lower than previously reported. Additionally, the age might be associated with the occurrence of OMLs in children in Xiangyun of Yunnan, China.

Artificial Synapses Based on Ferroelectric Schottky Barrier Field-Effect Transistors for Neuromorphic Applications.

Artificial synapses based on ferroelectric Schottky barrier field-effect transistors (FE-SBFETs) are experimentally demonstrated. The FE-SBFETs employ single-crystalline NiSi2 contacts with an atomically flat interface to Si and Hf0.5Zr0.5O2 ferroelectric layers on silicon-on-insulator substrates. The ferroelectric polarization switching dynamics gradually modulate the NiSi2/Si Schottky barriers and the potential of the channel, thus programming the device conductance with input voltage pulses. The short-term synaptic plasticity is characterized in terms of excitatory/inhibitory post-synaptic current (EPSC) and paired-pulse facilitation/depression. The EPSC amplitude shows a linear response to the amplitude of the pre-synaptic spike. Very low energy/spike consumption as small as ∼2 fJ is achieved, demonstrating high energy efficiency. Long-term potentiation/depression results show very high endurance and very small cycle-to-cycle variations (∼1%) after 105 pulse measurements. Furthermore, spike-timing-dependent plasticity is also emulated using the gate voltage pulse as the pre-synaptic spike and the drain voltage pulse as the post-synaptic spikes. These findings indicate that FE-SBFET synapses have high potential for future neuromorphic computing applications.

A Smart Robotic Walker With Intelligent Close-Proximity Interaction Capabilities for Elderly Mobility Safety.

The elderly population has rapidly increased in past years, bringing huge demands for elderly serving devices, especially for those with mobility impairment. Present assistant walkers designed for elderly users are primitive with limited user interactivity and intelligence. We propose a novel smart robotic walker that targets a convenient-to-use indoor walking aid for the elderly. The walker supports multiple modes of interactions through voice, gait or haptic touch, and allows intelligent control via learning-based methods to achieve mobility safety. Our design enables a flexible, initiative and reliable walker due to the following: (1) we take a hybrid approach by combining the conventional mobile robotic platform with the existing rollator design, to achieve a novel robotic system that fulfills expected functionalities; (2) our walker tracks users in front by detecting lower limb gait, while providing close-proximity walking safety support; (3) our walker can detect human intentions and predict emergency events, e.g., falling, by monitoring force pressure on a specially designed soft-robotic interface on the handle; (4) our walker performs reinforcement learning-based sound source localization to locate and navigate to the user based on his/her voice signals. Experiment results demonstrate the sturdy mechanical structure, the reliability of multiple novel interactions, and the efficiency of the intelligent control algorithms implemented. The demonstration video is available at: https://sites.google.com/view/smart-walker-hku.

Ectopic Parotid Main Duct on Buccal Mucosa: A Case Report and Literature Review.

Ectopic parotid main duct (Stensen's duct) orifice is a rare anatomic abnormality of the salivary gland. We report a case in which the patient's left Stensen's duct opened on the left buccal skin. After surgery to perform parotid duct transposition, the buccal fistula was closed, and the saliva secreted by the parotid gland could overflow smoothly through the new orifice throughout a 3-year follow-up period. We also performed a review of the current relevant data.

Phase evolution of ultra-thin Ni silicide films on CF4 plasma immersion ion implanted Si.

We present a systematic study on the effects of CF4 plasma immersion ion implantation (PIII) in Si on the phase evolution of ultra-thin Ni silicides. For 3 nm Ni, NiSi2 was formed on Si substrates with and without CF4 PIII at temperature as low as 400 °C. For 6 nm Ni, NiSi was formed on pure Si, while epitaxial NiSi2 was obtained on CF4 PIII Si. The incorporation of C and F atoms in the thin epitaxial NiSi2 significantly reduces the layer resistivity. Increasing the Ni thickness to 8 nm results in the formation of NiSi, where the thermal stability of NiSi, the NiSi/Si interface and Schottky contacts are significantly improved with CF4 PIII. We suggest that the interface energy is lowered by the F and C dopants present in the layer and at the interface, leading to phase evolution of the thin Ni silicide.

Comparison of EpCAMhighCD44+ cancer stem cells with EpCAMhighCD44- tumor cells in colon cancer by single-cell sequencing.

Cancer stem cells (CSCs) are considered to be responsible for tumorigenesis and cancer relapse. EpCAMhighCD44+ tumor cells are putative colorectal CSCs that express high levels of stem cell genes, while the EpCAMhighCD44- population mostly contains differentiated tumor cells (DTCs). This study aims to determine whether single CSC (EpCAMhighCD44+) and DTC (EpCAMhighCD44-) can be distinguished in terms of somatic copy number alterations (SCNAs). We applied fluorescence-activated cell sorting to isolate the CD45-EpCAMhighCD44+ and CD45-EpCAMhighCD44- populations from two primary colon tumors, on which low-coverage single-cell whole-genome sequencing (WGS) was then performed ∼0.1x depth. We compared the SCNAs of the CSCs and DTCs at single-cell resolution. In total, 47 qualified single cells of the two populations underwent WGS. The single-cell SCNA profiles showed that there were obvious SCNAs in both the CSCs and DTCs of each patient, and each patient had a specific copy number alteration pattern. Hierarchical clustering and correlation analysis both showed that the SCNA profiles of CSCs and DTCs from the same patient had similar SCNA pattern, while there were regional differences in the CSCs and DTCs in certain patient. SCNAs of CSCs in the same patient were highly reproducible. Our data suggest that major SCNAs occurred at an early stage and were inherited steadily. The similarity of ubiquitous SCNAs between the CSCs and DTCs might have arisen from lineage differentiation. CSCs from the same patient had reproducible SCNA profiles, indicating that gain or loss in certain chromosome is required for colon cancer development.

Transcriptional profiles of different states of cancer stem cells in triple-negative breast cancer.

Breast cancer stem cells (BCSCs) are thought to be responsible for tumor initiation, metastasis and relapse. Our group and others have described markers useful in isolating BCSCs just as aldehyde dehydrogenase positive (ALDH+) or CD24-CD44+. In fact, cells which simultaneously express both sets of markers have the highest tumor initiating capacity. Although the transcriptomic profile of cells expressing each BCSC marker alone has been reported, the profile of the most tumorigenic population expressing both sets of markers has not. Here we used the biomarker combination of ALDH and CD24/CD44 to sort four populations isolated from triple-negative breast cancer (TNBC) patient-derived xenografts, and performed whole-transcriptome sequencing on each population. We systematically compared the profiles of the three states of BCSCs (ALDH+CD24-CD44+, ALDH+non-CD24-CD44+ and ALDH-CD24-CD44+) to that of the differentiated tumor cells (ALDH-non-CD24-CD44+). For the first time, we compared the ALDH+CD24-CD44+ BCSCs with the other two BCSC populations. In ALDH+CD24-CD44+ BCSCs, we identified P4HA2, PTGR1 and RAB40B as potential prognostic markers, which were virtually related to the status of BCSCs and tumor growth in TNBC cells.

Multi-region and single-cell sequencing reveal variable genomic heterogeneity in rectal cancer.

BACKGROUND: Colorectal cancer is a heterogeneous group of malignancies with complex molecular subtypes. While colon cancer has been widely investigated, studies on rectal cancer are very limited. Here, we performed multi-region whole-exome sequencing and single-cell whole-genome sequencing to examine the genomic intratumor heterogeneity (ITH) of rectal tumors. METHODS: We sequenced nine tumor regions and 88 single cells from two rectal cancer patients with tumors of the same molecular classification and characterized their mutation profiles and somatic copy number alterations (SCNAs) at the multi-region and the single-cell levels. RESULTS: A variable extent of genomic heterogeneity was observed between the two patients, and the degree of ITH increased when analyzed on the single-cell level. We found that major SCNAs were early events in cancer development and inherited steadily. Single-cell sequencing revealed mutations and SCNAs which were hidden in bulk sequencing. In summary, we studied the ITH of rectal cancer at regional and single-cell resolution and demonstrated that variable heterogeneity existed in two patients. The mutational scenarios and SCNA profiles of two patients with treatment naïve from the same molecular subtype are quite different. CONCLUSIONS: Our results suggest each tumor possesses its own architecture, which may result in different diagnosis, prognosis, and drug responses. Remarkable ITH exists in the two patients we have studied, providing a preliminary impression of ITH in rectal cancer.

Live births after simultaneous avoidance of monogenic diseases and chromosome abnormality by next-generation sequencing with linkage analyses.

In vitro fertilization (IVF), preimplantation genetic diagnosis (PGD), and preimplantation genetic screening (PGS) help patients to select embryos free of monogenic diseases and aneuploidy (chromosome abnormality). Next-generation sequencing (NGS) methods, while experiencing a rapid cost reduction, have improved the precision of PGD/PGS. However, the precision of PGD has been limited by the false-positive and false-negative single-nucleotide variations (SNVs), which are not acceptable in IVF and can be circumvented by linkage analyses, such as short tandem repeats or karyomapping. It is noteworthy that existing methods of detecting SNV/copy number variation (CNV) and linkage analysis often require separate procedures for the same embryo. Here we report an NGS-based PGD/PGS procedure that can simultaneously detect a single-gene disorder and aneuploidy and is capable of linkage analysis in a cost-effective way. This method, called "mutated allele revealed by sequencing with aneuploidy and linkage analyses" (MARSALA), involves multiple annealing and looping-based amplification cycles (MALBAC) for single-cell whole-genome amplification. Aneuploidy is determined by CNVs, whereas SNVs associated with the monogenic diseases are detected by PCR amplification of the MALBAC product. The false-positive and -negative SNVs are avoided by an NGS-based linkage analysis. Two healthy babies, free of the monogenic diseases of their parents, were born after such embryo selection. The monogenic diseases originated from a single base mutation on the autosome and the X-chromosome of the disease-carrying father and mother, respectively.