Accelerating attribute-focused process and product development through the development and deployment of autonomous process analytical technology platform system.

Enabling real-time monitoring and control of the biomanufacturing processes through product quality insights continues to be an area of focus in the biopharmaceutical industry. The goal is to manufacture products with the desired quality attributes. To realize this rigorous attribute-focused Quality by Design approach, it is critical to support the development of processes that consistently deliver high-quality products and facilitate product commercialization. Time delays associated with offline analytical testing can limit the speed of process development. Thus, developing and deploying analytical technology is necessary to accelerate process development. In this study, we have developed the micro sequential injection process analyzer and the automatic assay preparation platform system. These innovations address the unmet need for an automatic, online, real-time sample acquisition and preparation platform system for in-process monitoring, control, and release of biopharmaceuticals. These systems can also be deployed in laboratory areas as an offline analytical system and on the manufacturing floor to enable rapid testing and release of products manufactured in a good manufacturing practice environment.

The prevalence of gastroesophageal reflux disease and laryngopharyngeal reflux in patients with dysphagia after radiotherapy for nasopharyngeal carcinoma.

BACKGROUND: The prevalence of gastroesophageal reflux disease (GERD) and laryngopharyngeal reflux (LPR) in post-irradiated patients with nasopharyngeal carcinoma (NPC) is unknown. MATERIALS AND METHODS: In a cross-sectional study, 31 NPC and 12 control patients completed questionnaires for GERD/LPR before esophageal manometry and 24-h pH monitoring. The DeMeester score and reflux finding score (RFS) were used to define GERD and LPR, respectively. Risk factors were identified. RESULTS: 51.6% of NPC and 8.3% of control patients, and 77.4% of NPC and 33% of control patients, were GERD-positive and LPR-positive, respectively. The GERD/LPR questionnaire failed to identify either condition in patients with NPC. No parameter differences in esophageal manometry or pneumonia incidence were noted between GERD/LPR-positive and GERD/LPR-negative patients. Post radiotherapy duration, high BMI, lack of chemotherapy, and dysphagia were positive risk factors for GERD/LPR. CONCLUSIONS: A high prevalence of GERD/LPR in patients with post-irradiated NPC exists, but reflux symptoms are inadequate for diagnosis.

Machine learning-coupled combinatorial mutagenesis enables resource-efficient engineering of CRISPR-Cas9 genome editor activities.

The genome-editing Cas9 protein uses multiple amino-acid residues to bind the target DNA. Considering only the residues in proximity to the target DNA as potential sites to optimise Cas9's activity, the number of combinatorial variants to screen through is too massive for a wet-lab experiment. Here we generate and cross-validate ten in silico and experimental datasets of multi-domain combinatorial mutagenesis libraries for Cas9 engineering, and demonstrate that a machine learning-coupled engineering approach reduces the experimental screening burden by as high as 95% while enriching top-performing variants by ∼7.5-fold in comparison to the null model. Using this approach and followed by structure-guided engineering, we identify the N888R/A889Q variant conferring increased editing activity on the protospacer adjacent motif-relaxed KKH variant of Cas9 nuclease from Staphylococcus aureus (KKH-SaCas9) and its derived base editor in human cells. Our work validates a readily applicable workflow to enable resource-efficient high-throughput engineering of genome editor's activity.

Oro-pharyngo-esophageal radionuclide scintigraphy predicts aspiration pneumonia risk and associated survival in post-irradiated nasopharyngeal carcinoma patients.

OBJECTIVE: To demonstrate that oro-pharyngo-esophageal radionuclide scintigraphy (OPERS) not only detects tracheobronchial aspiration after swallowing, but also quantifies the amount of aspiration and subsequent clearance. METHODS: Data collected between 2014 and 2019 were reviewed for aspiration pneumonia at 12 and 24-months after OPERS. The predictive value for aspiration pneumonia on flexible endoscopic evaluation of swallowing (FEES), videofluoroscopic swallowing study (VFSS), and OPERS, and the overall survival of patients with or without aspiration were determined. RESULTS: Thirty-seven patients treated with radiotherapy for nasopharyngeal carcinoma (NPC) were reviewed. The incidence of aspiration detected on FEES, VFSS, and OPERS was 78.4%, 66.7%, and 44.4%, respectively. Using VFSS as a gold standard, the sensitivity and specificity of OPERS for aspiration was 73.7% and 100%. The positive and negative predictive values for aspiration were 100% and 66.7%, respectively, with an overall accuracy of 82.8%. A history of aspiration pneumonia was one factor associated with a higher chance of subsequent aspiration pneumonia within 12 months (odds ratio: 15.5, 95% CI 1.67-145.8, p < .05) and 24 months (odds ratio: 23.8, 95% CI 3.69-152.89, p < .01) of the swallowing assessment. Aspiration detected by OPERS was a significant risk factor for future aspiration pneumonia at 12 and 24 months respectively. Significantly, better survival was associated with an absence of aspiration on OPERS only, but not on FEES or VFSS. CONCLUSION: OPERS predicts the safety of swallowing, the incidence of subsequent aspiration pneumonia, and the survival prognosis in post-irradiated NPC dysphagia patients. LEVEL OF EVIDENCE: 3.

High-fidelity KKH variant of Staphylococcus aureus Cas9 nucleases with improved base mismatch discrimination.

The Cas9 nuclease from Staphylococcus aureus (SaCas9) holds great potential for use in gene therapy, and variants with increased fidelity have been engineered. However, we find that existing variants have not reached the greatest accuracy to discriminate base mismatches and exhibited much reduced activity when their mutations were grafted onto the KKH mutant of SaCas9 for editing an expanded set of DNA targets. We performed structure-guided combinatorial mutagenesis to re-engineer KKH-SaCas9 with enhanced accuracy. We uncover that introducing a Y239H mutation on KKH-SaCas9's REC domain substantially reduces off-target edits while retaining high on-target activity when added to a set of mutations on REC and RuvC domains that lessen its interactions with the target DNA strand. The Y239H mutation is modelled to have removed an interaction from the REC domain with the guide RNA backbone in the guide RNA-DNA heteroduplex structure. We further confirmed the greatly improved genome-wide editing accuracy and single-base mismatch discrimination of our engineered variants, named KKH-SaCas9-SAV1 and SAV2, in human cells. In addition to generating broadly useful KKH-SaCas9 variants with unprecedented accuracy, our findings demonstrate the feasibility for multi-domain combinatorial mutagenesis on SaCas9's DNA- and guide RNA- interacting residues to optimize its editing fidelity.

A Combinatorial CRISPR-Cas9 Screen Identifies Ifenprodil as an Adjunct to Sorafenib for Liver Cancer Treatment.

Systematic testing of existing drugs and their combinations is an attractive strategy to exploit approved drugs for repurposing and identifying the best actionable treatment options. To expedite the search among many possible drug combinations, we designed a combinatorial CRISPR-Cas9 screen to inhibit druggable targets. Coblockade of the N-methyl-d-aspartate receptor (NMDAR) with targets of first-line kinase inhibitors reduced hepatocellular carcinoma (HCC) cell growth. Clinically, HCC patients with low NMDAR1 expression showed better survival. The clinically approved NMDAR antagonist ifenprodil synergized with sorafenib to induce the unfolded protein response, trigger cell-cycle arrest, downregulate genes associated with WNT signaling and stemness, and reduce self-renewal ability of HCC cells. In multiple HCC patient-derived organoids and human tumor xenograft models, the drug combination, but neither single drug alone, markedly reduced tumor-initiating cancer cell frequency. Because ifenprodil has an established safety history for its use as a vasodilator in humans, our findings support the repurposing of this drug as an adjunct for HCC treatment to improve clinical outcome and reduce tumor recurrence. These results also validate an approach for readily discovering actionable combinations for cancer therapy. SIGNIFICANCE: Combinatorial CRISPR-Cas9 screening identifies actionable targets for HCC therapy, uncovering the potential of combining the clinically approved drugs ifenprodil and sorafenib as a new effective treatment regimen.

Assessment of pharyngeal motor function using a novel velopharyngeal squeeze maneuver and a novel endoscopic pharyngeal contraction grade scale in patients with dysphagia after radiotherapy for nasopharyngeal carcinoma.

BACKGROUND: To investigate a novel velopharyngeal squeeze maneuver (VPSM) and novel endoscopic pharyngeal contraction grade (EPCG) scale for the evaluation of pharyngeal motor function. METHODS: During endoscopic examination of 77 post-irradiated nasopharyngeal carcinoma patients and control subjects, VPSM was rated and lateral pharyngeal wall movement graded with EPCG scale during swallowing. Pharyngeal constriction ratio (PCR) measured by videofluoroscopy was used for correlation. RESULTS: VPSM and EPCG scale showed almost perfect intra-rater and inter-rater reliability (Kappa: >0.90). VPSM was present in 61% of patients suggesting good pharyngeal motor function. VPSM was predictive of EPCG scale (Wald statistic = 29.99, p < 0.001). EPCG scale also correlated strongly with PCR (r: 0.812) and was predictive for aspiration (odds ratio: 22.14 [95% CI 5.01-97.89, p < 0.001]). CONCLUSIONS: VPSM and EPCG scale are two novel tools to assess pharyngeal motor function, and both correlate well with pharyngeal contractility and aspiration.

Extensible combinatorial CRISPR screening in mammalian cells.

The CRISPR-Cas system coupled with Combinatorial Genetics En Masse (CombiGEM) enables systematic analysis of high-order genetic perturbations that are important for understanding biological processes and discovering therapeutic target combinations. Here, we present detailed steps and technical considerations for building multiplexed guide RNA libraries and carrying out a combinatorial CRISPR screen in mammalian cells. We also present an analytical pipeline, CombiPIPE, for mapping two- and three-way genetic interactions. For complete details on the use and execution of this protocol, please refer to Zhou et al. (2020).

Development and pilot evaluation of the Cognition domain of the Hong Kong Comprehensive Assessment Scales for Toddlers.

Purpose: To describe the development of the Cognition domain of the Hong Kong Comprehensive Assessment Scales for Toddlers (HKCAS-T).Methods: Participants included 345 toddlers aged 18-41 months, with 258 recruited from Maternal and Child Health Centers (MCHCs) and 87 with cognitive delay recruited from Child Assessment Centers (CACs). They were individually administered the 83-item pilot version by medical practitioners or educational psychologists between 2017 and 2019 in MCHCs and CACs in Hong Kong.Results: Rasch analysis results supported the unidimensionality of the pilot version, after removing six items. Analysis of covariance results indicated that both the 83-item version and the 77-item version could differentiate between children of different age groups, and children with typical development from children with cognitive delay. Internal consistency and interrater reliability were 0.90 or above.Conclusions: The Cognition domain of the HKCAS-T is a promising developmental assessment tool for the assessment of toddlers. Cognition assessment, preschool, Chinese.

A Three-Way Combinatorial CRISPR Screen for Analyzing Interactions among Druggable Targets.

We present a CRISPR-based multi-gene knockout screening system and toolkits for extensible assembly of barcoded high-order combinatorial guide RNA libraries en masse. We apply this system for systematically identifying not only pairwise but also three-way synergistic therapeutic target combinations and successfully validate double- and triple-combination regimens for suppression of cancer cell growth and protection against Parkinson's disease-associated toxicity. This system overcomes the practical challenges of experimenting on a large number of high-order genetic and drug combinations and can be applied to uncover the rare synergistic interactions between druggable targets.

Pearls of experience for safe and efficient hospital practices in otorhinolaryngology-head and neck surgery in Hong Kong during the 2019 novel coronavirus disease (COVID-19) pandemic.

The 2019 novel coronavirus disease (COVID-19) epidemic originated in Wuhan, China and spread rapidly worldwide, leading the World Health Organization to declare an official global COVID-19 pandemic in March 2020. In Hong Kong, clinicians and other healthcare personnel collaborated closely to combat the outbreak of COVID-19 and minimize the cross-transmission of disease among hospital staff members. In the field of otorhinolaryngology-head and neck surgery (OHNS) and its various subspecialties, contingency plans were required for patient bookings in outpatient clinics, surgeries in operating rooms, protocols in wards and other services. Infected patients may shed severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) particles into their environments via body secretions. Therefore, otolaryngologists and other healthcare personnel in this specialty face a high risk of contracting COVID-19 and must remain vigilant when performing examinations and procedures involving the nose and throat. In this article, we share our experiences of the planning and logistics undertaken to provide safe and efficient OHNS practices over the last 2 months, during the COVID-19 pandemic. We hope that our experiences will serve as pearls for otolaryngologists and other healthcare personnel working in institutes that serve large numbers of patients every day, particularly with regard to the sharing of clinical and administrative tasks during the COVID-19 pandemic.

Management of dysphagia in the patient with head and neck cancer during COVID-19 pandemic: Practical strategy.

The global pandemic of 2019 novel coronavirus disease (COVID-19) has tremendously altered routine medical service provision and imposed unprecedented challenges to the health care system. This impacts patients with dysphagia complications caused by head and neck cancers. As this pandemic of COVID-19 may last longer than severe acute respiratory syndrome (SARS) in 2003, a practical workflow for managing dysphagia is crucial to ensure a safe and efficient practice to patients and health care personnel. This document provides clinical practice guidelines based on available evidence to date to balance the risks of SARS-CoV-2 exposure with the risks associated with dysphagia. Critical considerations include reserving instrumental assessments for urgent cases only, optimizing the noninstrumental swallowing evaluation, appropriate use of personal protective equipment (PPE), and use of telehealth when appropriate. Despite significant limitations in clinical service provision during the pandemic of COVID-19, a safe and reasonable dysphagia care pathway can still be implemented with modifications of setup and application of newer technologies.

Publisher Correction: Combinatorial mutagenesis en masse optimizes the genome editing activities of SpCas9.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Combinatorial mutagenesis en masse optimizes the genome editing activities of SpCas9.

The combined effect of multiple mutations on protein function is hard to predict; thus, the ability to functionally assess a vast number of protein sequence variants would be practically useful for protein engineering. Here we present a high-throughput platform that enables scalable assembly and parallel characterization of barcoded protein variants with combinatorial modifications. We demonstrate this platform, which we name CombiSEAL, by systematically characterizing a library of 948 combination mutants of the widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease to optimize its genome-editing activity in human cells. The ease with which the editing activities of the pool of SpCas9 variants can be assessed at multiple on- and off-target sites accelerates the identification of optimized variants and facilitates the study of mutational epistasis. We successfully identify Opti-SpCas9, which possesses enhanced editing specificity without sacrificing potency and broad targeting range. This platform is broadly applicable for engineering proteins through combinatorial modifications en masse.

Does Low Dose Angiotensin Converting Enzyme Inhibitor Prevent Pneumonia in Older People With Neurologic Dysphagia--A Randomized Placebo-Controlled Trial.

OBJECTIVE: To examine if angiotensin converting enzyme inhibitor reduces the risk of pneumonia in older patients on tube-feeding because of dysphagia from cerebrovascular diseases. DESIGN: Randomized placebo-controlled trial. SETTING: Acute and subacute geriatrics units, speech therapists' clinic, and nursing home. PARTICIPANTS: Older patients on tube-feeding for >2 weeks because of dysphagia secondary to cerebrovascular diseases. INTERVENTION: Participants were randomized to lisinopril 2.5 mg or placebo once daily for 26 weeks. MEASUREMENTS: Participants were followed up at weeks 12 and 26. The primary outcome was the incidence rate of pneumonia as determined by pneumonic changes on x-ray and clinical criteria. The secondary outcomes were mortality rate and swallowing ability as defined by the Royal Brisbane Hospital Outcome Measure for Swallowing at week 12. RESULTS: A total of 93 older patients were randomized. In interim analysis, 71 completed the trial, whereas 15 had dropped out. Among those who had completed the trial, odds ratio (OR) for death was significantly higher in the intervention group (unadjusted OR 2.94, P = .030; fully adjusted OR 7.79, P = .018). There was no difference in the incidence of pneumonia or fatal pneumonia in the 2 groups. The intervention group had a marginally better swallowing function at week 12 (Royal Brisbane Hospital Outcome Measure for Swallowing score: 4.2 ± 1.5 in intervention group, 3.5 ± 1.5 in placebo group, P = .053). As a result of the interim finding on mortality, the trial was prematurely terminated with 7 participants still in the trial. CONCLUSIONS: Low dose lisinopril given to older tube-fed patients with neurologic dysphagia resulted in increased mortality, although swallowing function showed marginal improvement. ACE inhibitors did not prevent pneumonia in older patients with neurologic dysphagia and might increase mortality.

The joint effects of risk status, gender, early literacy and cognitive skills on the presence of dyslexia among a group of high-risk Chinese children.

This study sought to examine factors that are predictive of future developmental dyslexia among a group of 5-year-old Chinese children at risk for dyslexia, including 62 children with a sibling who had been previously diagnosed with dyslexia and 52 children who manifested clinical at-risk factors in aspects of language according to testing by paediatricians. The age-5 performances on various literacy and cognitive tasks, gender and group status (familial risk or language delayed) were used to predict developmental dyslexia 2 years later using logistic regression analysis. Results showed that greater risk of dyslexia was related to slower rapid automatized naming, lower scores on morphological awareness, Chinese character recognition and English letter naming, and gender (boys had more risk). Three logistic equations were generated for estimating individual risk of dyslexia. The strongest models were those that included all print-related variables (including speeded number naming, character recognition and letter identification) and gender, with about 70% accuracy or above. Early identification of those Chinese children at risk for dyslexia can facilitate better dyslexia risk management.

Early predictors of dyslexia in Chinese children: familial history of dyslexia, language delay, and cognitive profiles.

BACKGROUND: This work tested the rates at which Chinese children with either language delay or familial history of dyslexia at age 5 manifested dyslexia at age 7, identified which cognitive skills at age 5 best distinguished children with and without dyslexia at age 7, and examined how these early abilities predicted subsequent literacy skills. METHOD: Forty-seven at-risk children (21 who were initially language delayed and 26 with familial risk) and 47 control children matched on age, IQ, and mothers' education were tested on syllable awareness, tone detection, rapid automatized naming, visual skill, morphological awareness, and word reading at age 5 and subsequently tested for dyslexia on a standard Hong Kong measure at age 7. RESULTS: Of those with an early language delay, 62% subsequently manifested dyslexia; for those with familial risk, the rate of dyslexia was 50%. Those with dyslexia were best distinguished from those without dyslexia by the age-5 measures of morphological awareness, rapid automatized naming, and word reading itself; other measures did not distinguish the groups. In a combined regression analysis across all participants, morphological awareness uniquely explained word reading accuracy and rapid automatized naming uniquely explained timed word reading at age 7, with all other measures statistically controlled. Separate stepwise regression analyses by group indicated that visual skill uniquely explained subsequent literacy skills in the at-risk group only, whereas tone and syllable awareness were unique predictors of literacy skills in the control group only. CONCLUSIONS: Both early language delay and familial risk strongly overlap with subsequent dyslexia in Chinese children. Overall, rapid automatized naming and morphological awareness are relatively strong correlates of developmental dyslexia in Chinese; visual skill and phonological awareness may also be uniquely associated with subsequent literacy development in at-risk and typically developing children, respectively.

The cognitive profile of Chinese children with mathematics difficulties.

This study examined how four domain-specific skills (arithmetic procedural skills, number fact retrieval, place value concept, and number sense) and two domain-general processing skills (working memory and processing speed) may account for Chinese children's mathematics learning difficulties. Children with mathematics difficulties (MD) of two age groups (7-8 and 9-11 years) were compared with age-matched typically achieving children. For both age groups, children with MD performed significantly worse than their age-matched controls on all of the domain-specific and domain-general measures. Further analyses revealed that the MD children with literacy difficulties (MD/RD group) performed the worst on all of the measures, whereas the MD-only group was significantly outperformed by the controls on the four domain-specific measures and verbal working memory. Stepwise discriminant analyses showed that both number fact retrieval and place value concept were significant factors differentiating the MD and non-MD children. To conclude, deficits in domain-specific skills, especially those of number fact retrieval and place value understanding, characterize the profile of Chinese children with MD.

Differential expression of tissue repair genes in the pathogenesis of chronic obstructive pulmonary disease.

RATIONALE: The airflow limitation that defines severity of chronic obstructive pulmonary disease (COPD) is caused by a combination of small airway obstruction and emphysematous lung destruction. OBJECTIVES: To examine the hypothesis that small airway obstructive and emphysematous destructive lesions are produced by differential expression of genes associated with tissue repair. METHODS: The expression of 54 genes associated with repair of repetitively damaged tissue was measured in 136 paired samples of small bronchioles and surrounding lung tissue separated by laser capture microdissection. These samples were collected from 63 patients at different levels of disease severity who required surgery for either lung cancer or lung transplantation for very severe COPD. Gene expression was measured by quantitative polymerase chain reaction in these paired samples and compared with the FEV(1) by linear regression analysis. MEASUREMENTS AND MAIN RESULTS: After corrections for false discovery rates, only 2 of 10 genes (serpin peptidase inhibitor/plasminogen activator inhibitor member 2 and matrix metalloproteinase [MMP] 10) increased, whereas 8 (MMP2, integrin-alpha1, vascular endothelial growth factor, a disintegrin and metallopeptidase domain 33, scatter factor/hepatocyte growth factor, tissue inhibitor of matrix metalloproteinase-2, fibronectin, and collagen 3alpha1) decreased in small airways in association with FEV(1). In contrast, 8/12 genes (early growth response factor 1, MMP1, MMP9, MMP10, plasminogen activator urokinase, plasminogen activator urokinase receptor, tumor necrosis factor, and IL13) increased and 4/12 (MMP2, tissue inhibitor of matrix metalloproteinase-1, collagen 1alpha1, and transforming growth factor-beta3) decreased in the surrounding lung tissue in association with progression of COPD. CONCLUSIONS: The progression of COPD is associated with the differential expression of a cluster of genes that favor the degradation of the tissue surrounding the small conducting airways.

Early swallowing problems in a cohort of patients with nasopharyngeal carcinoma: Symptomatology and videofluoroscopic findings.

OBJECTIVE: To study the incidence and the degree of swallowing dysfunction in patients with nasopharyngeal carcinoma (NPC) who underwent radiation therapy treatment. INSTITUTION: The study was conducted in the Prince of Wales Hospital, a tertiary teaching hospital of the Chinese University of Hong Kong. MATERIALS AND METHODS: From October 1999 to July 2001, a cohort of 20 consecutive patients with newly diagnosed NPC was prospectively studied. Questions about symptoms, including swallowing functions, were asked, and head and neck examination including oromotor examination was performed in the subjects before radiation therapy. All patients were subjected to videofluoroscopy (VFSS) to assess their swallowing function. Abnormalities were scored if they were present on two of three swallow attempts. The patients were reassessed at 6 months and 12 months after radiotherapy by symptom assessment and VFSS. RESULTS: There were 14 male and 6 female patients. The mean age was 43.9 years. Nine patients had early (stage I and II) disease, whereas 11 patients had advanced (stage III and IV) disease. Nine patients were treated by radiation therapy only and 11 patients by concurrent chemoirradiation. Ninety-five percent of the subjects had subjective dysphagia at 6 and 12 months after radiation therapy. Ninety percent had xerostomia, and 80% had to avoid certain foods at 12 months postradiation therapy. All subjects had to alternate solid food with fluid intake to facilitate swallowing. An average reduction of jaw movement by 1 cm was noted. A large proportion of patients had stasis of food in the pharynx (100% in valleculae and 60% in pyriform fossae) and impaired pharyngeal peristalsis (60%). One quarter of patients had laryngeal penetration. CONCLUSIONS: Subjective swallowing difficulties were common in patients in the early follow-up period after radiation therapy for NPC according to questionnaire assessment. An objective swallowing study revealed that swallowing dysfunction was persistent 12 months after radiation therapy.