Near-infrared room-temperature phosphorescence was achieved by employing iodine substituted Bodipy into amorphous polymers. The self-healable gels were also obtained with the incorporation of a crosslinker and quadruple hydrogen bond based moieties. N,N′-methylenebisacrylamide (MBAA). The mechanical properties of the gels were measured using a rheometer and the results showed that the gels displayed fast self-healing ability due to the strong quadruple hydrogen bonding between UPy moieties.Research on pure organic room-temperature phosphorescent (RTP) materials has made great advances but near-infrared (NIR) RTP emitting materials are still rare. Novel amorphous acrylamide copolymers containing iodine substituted borondipyrromethene (Bodipy) were prepared to obtain strong absorption in the visible region and moderate RTP in the NIR region with much larger Stokes shift than the fluorescence emission of traditional Bodipy dyes. Expensive metals and crystallization were left out to avoid biotoxicity and strict preparation conditions. Monoiodo and diiodo-Bodipy derivatives were both designed to study the substitution effect of iodine atoms. Photophysical properties, phosphorescence quantum yield and lifetime were characterized. Gels with NIR RTP emission were facilely prepared with the incorporation of ureidopyrimidone (UPy) and  Room-temperature phosphorescence (RTP) emitting materials have been one of the most extensively investigated luminescent materials in recent years due to their distinctive photophysical properties and wide applications in various fields including bioimaging,N,N′-methylenebisacrylamide (MBAA) and ureidopyrimidone (UPy) moieties into the polymerization process, self-healable NIR RTP emitting gels were obtained. UPy moieties can form stable dimers through quadruple hydrogen bonding which is extremely strong with an outstanding association constant (around 6 × 107 M–1 in CHCl3).Herein, two pure organic NIR RTP emitting materials in the amorphous state are facilely prepared by radical binary copolymerization of acrylamide and iodine-substituted Bodipy dyes . The heaCopolymerizing acrylamide and pure organic phosphors has been demonstrated to be an efficient and simple way to engender RTP emission. The rigid matrix of polyacrylamide can effectively immobilize the phosphors and inhibit the nonradiative transition process. Different colours of RTP including blue-purple, green, and orange have been achieved by employing several different phosphors.Spectral characteristics of the polymer monomers (1IBDP and 2IBDP) were studied using UV-Vis absorption spectra and fluorescence spectra. UV-Vis absorption spectra in diluted dichloromethane show that both 1IBDP and 2IBDP have strong sharp absorption peaks over 500 nm. Upon excitation at their maximum absorption wavelengths (518 nm and 535 nm), 1IBDP and 2IBDP showed fluorescence emission peaks at 535 nm and 555 nm, respectively Fig. S1. SimilarAfter copolymerization with acrylamide, amorphous powders p-1IBDP and p-2IBDP were obtained. UV-Vis absorption spectra of the polymers in the solid state are slightly different from the polymer monomers in diluted solutions . P-1IBDPThe heavy atom effect generally plays a key role in promoting ISC by enhancing spin–orbital coupling and inducing a triplet excited state. In this research work, p-1IBDP, p-2IBDP and a reference polymer p-BDP with no iodine substitution Scheme S2 were des1–T1 ISC caused by the heavy atom effect. All the photophysical properties, including phosphorescence quantum yields, are listed in With more iodine substituents on the Bodipy core, p-2IBDP exhibits weaker fluorescence and stronger phosphorescence . It is c2O mixed solvent was utilized since the acrylamide polymers were only soluble in water and insoluble in most organic solvents. With the existence of water, the rigid matrix of polyacrylamide and the hydrogen bonding inside the polymeric chains will be broken, which will enhance the molecular vibration and lead to oxygen quenching effects. Both P-1IBDP and p-2IBDP were suspended in the mixed solvent with different water fractions. With the increase of water fraction in the mixed solvent, RTP of the two polymers decreased remarkably and was totally quenched when water fraction reached 0.2%  analysis was performed to verify the amorphous state of p-1IBDP and p-2IBDP. Similar to the pure polyacrylamide,via quadruple hydrogen bonding interaction and have been utilized to prepare self-healable polymer gels.G′) are distinctly larger than loss modulus (G′′), verifying the formation of the gels. Both g-2IBDP and the healed gel show constant G′ and G′′ from 0.1% to 100%. With the applied strain increasing up to 1000%, no crossover point is observed between G′ and G′′, suggesting that the gels could withstand large deformations from damage (ω) dependence of G′ and G′′ also exhibits typical elastic gel behaviour of the samples  composed of acrylamide and iodine substituted Bodipy dyes were designed. The preparation of the polymers is facile with high yields. Both of the polymers have strong absorption in the visible region and RTP emissions at 770 nm in the amorphous state, which is the first report of pure organic RTP emission in the NIR region. The phosphors are immobilized and isolated from oxygen molecules by the rigid polymer matrix to reduce nonradiative transition. The number of iodine substitutions on the Bodipy core remarkably influences the fluorescence and RTP emission intensity. It has been proved that more iodine substitution leads to more efficient ISC, along with weaker fluorescence and stronger phosphorescence. Polymer gels with NIR RTP emission were also successfully obtained with the addition of MBAA and UPy moieties. The gels exhibit fast self-healing capacity because of the strong quadruple hydrogen bonding between UPy moieties. This study would be beneficial for the future design of multifunctional and smart RTP materials with potential applications in various fields.The authors declare no competing financial interests.Supplementary informationClick here for additional data file.


Much research has been carried out on the bacterial bioremediation of soil contaminated with petroleum hydrocarbons and toxic metals but much less is known about the potential of fungi in sites that are co-contaminated with both classes of pollutants. This article documents the roles of fungi in soil polluted with both petroleum hydrocarbons and toxic metals as well as the mechanisms involved in the biotransformation of such substances. Soil characteristics  and intracellular or excreted extracellular enzymes and metabolites are crucial factors which affect the efficiency of combined pollutant transformations. At present, bioremediation of soil co-contaminated with petroleum hydrocarbons and toxic metals is mostly focused on the removal, detoxification, or degradation efficiency of single or composite pollutants of each type. Little research has been carried out on the metabolism of fungi in response to complex pollutant stress. To overcome current bottlenecks in understanding fungal bioremediation, the potential of new approaches, e.g., gradient diffusion film technology (DGT) and metabolomics, is also discussed.Fungi play important roles in soil co-contaminated with TPH and toxic metals.• Soil characteristics, enzymes, and metabolites are major factors in bioremediation.• DGT and metabolomics can be applied to overcome current bottlenecks.•  With the accelerating pace of industrialization and urbanization, soil contamination has become a critical worldwide concern because of the threat to natural ecosystems and human health and much research has been carried out on innovative and cost-effective remediation technologies  resulting in enzyme dysfunction (Sandrin and Maier Remediation of soil co-contaminated with organic and inorganic pollutants is a complex problem as these two pollutant classes need to be treated differently Gadd . The preFungi are ubiquitous chemoorganotrophic (heterotrophic) organisms Gadd , and areAspergillus and Penicillium spp., have been investigated for the degradation of aliphatic hydrocarbons, chlorophenols, and polycyclic aromatic hydrocarbons, with the organic pollutants serving as carbon and energy sources Aspergillsms Gadd . Some fusms Gadd . Fusariu−1 Cd2+ inhibited the dechlorination of trichloroaniline (TCA) while 0.2-mg L−1 Cd2+ was necessary in an organic-dominated soil, which correlated with the metal-binding capacity of the organic material  (pH = 1.7–3.2) and Zn3(PO4)2∙4H2O (pH = 3.2–14) were the main mineral phases over different pH ranges   are promising candidates for the immobilization of toxic metals because the mechanism involved is associated with urea degradation. Such fungi grown in urea-containing medium hydrolyze urea producing ammonia and free carbonate which results in the precipitation of metals as carbonates, e.g., BaCO3, CdCO3, CoCO3, Cu2(OH)2CO3, La2(CO3)3, and NiCO3 , which conceal the dynamic changes in biodiversity or chemical speciation that may occur in contaminated soil. To overcome these difficulties, new technology, including microbial metabolomics (Dombrowski et al.


Pre-operative treatment planning in head and neck squamous cell carcinoma (HNSCC) is mainly dictated by clinical staging, which has major shortcomings. Histologic grading is irrelevant due to its lack of prognostic impact. Recently, a novel grading termed Cellular Dissociation Grade (CDG) based on Tumour Budding and Cell Nest Size was shown to be highly prognostic for resected HNSCC. We aimed to probe the predictive and prognostic impact of CDG in the pre-operative biopsies of HNSCC.n = 160 pre-therapeutic biopsies from patients who received standardised treatment following German guidelines, and correlated the results with pre- and post-therapeutic staging data and clinical outcome.We evaluated CDG in p < 0.001). Hazard ratio for disease-specific survival was 6.1 (11.1) for nG2 (nG3) compared with nG1 tumours.Pre-operative CDG was highly predictive of post-operative tumour stage, including the prediction of occult lymph node metastasis. Uni- and multivariate analysis revealed CDG to be an independent prognosticator of overall, disease-specific and disease-free survival (CDG is a strong outcome predictor in the pre-treatment scenario of HNSCC and identifies patients with nodal-negative disease. CDG is a purely histology-based prognosticator in the pre-therapeutic setting that supplements clinical staging and may aide therapeutic stratification of HNSCC patients. Human Papilloma Virus (HPV)— negative cases are particularly located within the oral cavity, hypopharynx and larynx.3 Overall, the prognosis of HNSCC, especially of HPV-negative cases, is unfavourable with a 5-year survival of patients on average below 50%.5 To date, tumour stage according to clinical and pathological TNM and UICC classification provides the most important prognostic information for patient outcome.5 However, pre-operative clinical assessment of tumour size (cT stage) and nodal involvement (cN stage) is discordant from pathological pT- and pN stage in nearly half of cases.6 Estimates assume that in early-stage oral squamous cell carcinoma (SCC) without clinically or radiologically apparent nodal metastasis 20–40% of patients contain occult metastasis at the time of diagnosis.9 As accurate routine biomarkers besides clinical staging for detection of such occult metastasis are still lacking, it is a matter of an ongoing debate which patients could be treated adequately and sufficiently without neck dissection in order to avoid overtreatment and in which patients to perform neck dissection in order to detect clinically non-apparent nodal metastasis and therewith improve prognosis.12Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers worldwide with ~500.000 new cases annually.17 In an effort to resolve this issue, our group recently proposed a new histopathological grading scheme termed Cellular Dissociation Grade (CDG) for oral, hypopharyngeal and laryngeal SCC, which is based on the parameters Tumour Budding and Cell Nest Size. These parameters describe the extent of Cellular Dissociation of a given cancer, both from a quantitative (Tumour Budding) and qualitative (Cell Nest Size) angle. CDG has not only been shown to be highly prognostic in other squamous cell carcinomas entities like oesophageal or cervical SCC but was also recently demonstrated to work nicely on pre-therapeutic biopsies of oesophageal SCC.Currently, histopathologic grading does not play a major role in clinical decision making as current histopathological grading schemes according to the World Health Organization (WHO) possess major shortcomings regarding prognostic patient stratification and reproducibility.19 However, up to date, pre-operative grading in HNSCC is almost meaningless as conventional grading according to the WHO is neither correlated with post-operative tumour stage nor with patient outcome,17 and is also known to be highly discordant when biopsies and corresponding resection specimen are compared.21Biopsies are the mainstay of pre-operative diagnostics in many cancer types, including HNSCC. Pre-therapeutic biopsies are not only necessary to establish a diagnosis but offer the possibility to determine histological subtypes, differentiation and potential further prognostic/predictive information to identify biologically aggressive tumours or to predict treatment response. Therewith, histopathological grading in biopsy specimen guides choice of therapies, including e.g., neoadjuvant systemic treatment versus primary resection or watchful waiting strategies in entities as endometrial cancer and prostate cancer.n = 160 patients with HNSCC located in the oral cavity, larynx and hypopharynx. We analysed concordance of CDG between biopsy and resection specimens and correlated CDG with pre-operative clinical staging , post-operative pathological staging  and with patient outcome , disease-specific (DSS) and disease-free survival (DFS)).Based on the above said, major questions remain. Can the high prognostic value of CDG be transferred to small biopsy specimens in HNSCC without losing its prognostic impact concerning patient survival? Can CDG potentially serve as a biomarker for prediction of occult metastasis in cN0 necks in clinically early-stage HNSCC? How is the concordance of CDG when biopsies and corresponding resection specimens are compared? Aiming to answer these questions, we investigated Tumour Budding and Cell Nest Size and CDG derived from these factors in pre-operative biopsies and resection specimen of 22 Clinical and pathological T-stage, N-stage and UICC stage, which were available for 100/160 (62.5%), were documented according to the TNM classification of malignant tumours.23 Distribution of clinicopathological parameters is given in Tables Included in our study were 160 patients with HNSCC of the larynx , the hypopharynx and the oral cavity from whom pre-operative biopsies and resection specimen were prospectively collected. All patients were treated in curative intent between 2004 and 2015 at Klinikum Rechts der Isar of the Technical University of Munich, Germany. All patients received a complete surgical resection of their primary tumours followed by a neck dissection in combination with radiotherapy (65–70 Gy) or radio-chemotherapy if indicated  according to German guidelines. Exclusion criteria were distant metastasis at the time of diagnosis (cM1 stage), neoadjuvant treatment with radio- and/or chemotherapy and pathological diagnosis upon fresh-frozen tissue sections. Male patients (129/160 (80.6%)) were in the majority compared with female counterparts (31/160 (19.4%)). Mean age at diagnosis was 60.6 years (range: 40.0–87.0 years). Mean follow-up time of patients alive at the endpoint of analysis was 53.5 months, mean survival time of deceased patients 26.9 months. During follow-up, 70/160 (43.8%) of patients died and 65/160 (40.6%) suffered from  relapse. Pre-operative clinical staging was performed by clinical examination, ultrasound and/or CT-scan according to the German guidelines.24 In case of discordance between biopsy and resection specimen, both biopsy and resection specimen were comparatively re-evaluated. HNSCC were categorised according to the current WHO classification criteria of tumours of head and neck into non-keratinising, keratinising and basaloid.3Biopsy and resection specimen from all 160 patients were available. Full block haematoxylin and eosin (H&E)-stained slides of biopsy specimen were evaluated simultaneously by experienced pathologists  blinded to clinicopathological data and follow-up using an Olympus BX43 microscope with a field diameter of 0.55 mm (0.24 mm²). Resection specimen had previously been evaluated in the context of two recently published studies.25 In short, Tumour Budding was defined as “branching” of small tumour nests harbouring <5 tumour cells into the stroma/parenchyma,27 and was assessed in one high-power-field (HPF) in biopsies and in ten (continuous) HPFs in resection specimen in areas showing maximal budding activity. The slight modification of the evaluated tumour area (one HPF in biopsies versus ten HPFs in resection specimen) was necessary due to the limited tissue availability in biopsy samples. Low budding activity was defined as 1–4 buds in one HPF (biopsies) and as 1–14 budding nests in ten HPFs (resections), high budding activity was defined as ≥5 in one HPF (biopsies) and as ≥15 budding nests in ten HPFs (resections). Overall Cell Nest Size, a parameter additionally measuring the dissociative growth capability of an individual tumour from a qualitative point of view, was evaluated in tumour area containing smallest tumour cell nests. Tumour cell nests, defined as clustered tumour cells surrounded by tumour stroma, were classified according the size of the smallest invasive cell nest as follows: > 15 tumour cells = large nests, 5–15 tumour cells = intermediate nests, 2–4 tumour cells = small nests, discohesive tumour cells without nested architecture = single-cell invasion. The criteria for budding activity and cell nest size evaluation applied were similar to already established algorithms for the evaluation of CDG in biopsies and resection specimen. According to our previously proposed grading system,25 scores for budding activity  and cell nest size  were assigned in order to obtain the CDG. After summarising both scores into a sum score from 2 to 7, HNSCC were graded into well cohesive , moderately cohesive  and poorly cohesive  according to the CDG.Tumour Budding and Cell Nest Size and subsequent CDG were evaluated as described before in detail.2 test as well as Χ2 test for trends and Fisher’s exact test. Cohen’s kappa was used to assess concordance between clinical and pathological staging parameters as well as to assess concordance between histopathological parameters in biopsies and resection specimen. Survival probabilities were plotted with the Kaplan–Meier method, a log-rank test was used to probe for the significance of differences in survival. Multivariate survival analysis was performed with the Cox proportional hazard model. P-values ≤ 0.05 were considered significant. All statistical tests were performed two-sided, and a significance level of 5% was used.Associations of grouped morphological characteristics with clinicopathological parameters were calculated with ΧThe majority of HNSCC were keratinising tumours , whereas non-keratinising cancers  and basaloid cancers  were less frequent. No tumour budding activity (per HPF) was detected in 34/160 (21.3%), while low budding activity was observed in 88/160 (55.0%) of cases and high budding activity in 38/160 (23.7%) of cases. Analysis of the Cell Nest Size revealed large cell nests in 16/160 cases (10.0%), intermediate nests in 18/160 cases (11.3%) and small cell nests in 55/160 cases (34.3%). In total, 71/160 cases (44.4%) showed infiltrative singular tumour cells, and were therefore classified as single-cell invasion. Addition of scores obtained for budding activity (scores 1–3) and cell nest size (scores 1–4) resulted in a sum scores 2–7 for each case, which was assigned to the final CDG  as described in the Methods section. While 34/160 (21.3%) HNSCC were well differentiated (nG1), 89/160 (55.6%) were moderately differentiated (nG2) and 37/160 (23.1%) poorly differentiated  cases. The corresponding Kappa value was κ = 0.87. Concerning Cell Nest Size, concordance rate was considerably lower with 102/160 (63.8%) HNSCC showing the same cell nest size in biopsy and resection specimen . Analysis of concordance rate between assigned grades resulted in 147/160 (93.1%) HNSCC with identical histopathological grade . Of the 13 cases with discordant grading, which was confirmed upon re-evaluation of biopsy and resection specimen, 12 showed an upgrading whereas only one case was downgraded from nG2 to nG1  and cN (p = 0.010) stage with single-cell invasion being more frequently detected in HNSCC patients with higher clinical stages. No further significant correlation of either budding activity or overall grading with clinical staging parameters were observed (Table Cell nest size was significantly correlated with cT (ed Table .p < 0.005). This was mirrored by correlation of moderate (nG2) and poor (nG3) differentiation with higher post-operative stages  HNSCC . In total, 14 cases received an upstaging and 22 a downstaging after pathological evaluation of the resection specimen. Respective values for nodal stage and UICC stage were as follows: discordance rate cN vs. pN 32/100 ; pre-operative UICC vs. post-operative UICC 33/100 . Upstaging: nodal stage 12 cases; UICC stage 14 cases. Downstaging: nodal stage 20 cases; UICC stage 19 cases (Supplementary Table n = 40) showed occult metastases detected by pathological evaluation of neck dissection specimen in 8/40 (20.0%) cases. Histopathological grading of all of these cases was nG2/3, whereas in none of the nG1 cases presence of lymph node metastases was observed. This result indicated a positive predictive value PPV = 100% for nG1 grading to predict nodal negativity upon pathological work-up in cN0 necks. Association of grading with post-operative nodal stage is summarised in Supplementary Table HNSCC patients with nodal-negative cervical lymph nodes upon clinical staging procedures  with HNSCC with high budding activity revealing the shortest mean OS. In analogy, Cell Nest Size was strongly correlated with OS, DSS and DFS . Cases with single-cell invasion and small cell nests suffered from earlier deaths and tumour recurrences compared with their counterparts   Table . While mp = 0.002; multivariate analysis including pUICC).In order to prove that the prognostic impact of the novel grading system derived from biopsy specimens is independent from clinicopathological data and tumour staging, we performed a multivariate analysis by applying a cox regression model incorporating pre-operative, respectively, post-operative staging parameters (cUICC/pUICC), gender and age and the CDG. Multivariate analysis confirmed the independent prognostic value of the novel grading concerning patient survival  or qualitative (Cell Nest Size) point of view. Considering the above discussed potential major prognostic and predictive implications of the data derived early in the diagnostic process from biopsy specimens, we aimed to probe if the prognostic value of the CDG is retained when assessed in pre-therapeutic biopsies of patients suffering from HNSCC. In addition, we analysed the value of the pre-operative CDG for the prediction of post-operative tumour stage, including the presence of occult lymph node metastasis in clinically and radiologically negative cN0 necks.We recently proposed a novel grading algorithm in HNSCC with high prognostic impact21 In addition, the Kappa value κ = 0.89 for the corresponding biopsy and resection specimen grading with 94% concordantly graded HNSCC proves the representativity of the biopsy grading results for the overall tumour configuration. Of 160 HNSCC, an upgrading from biopsy to resection specimen was only observed in ten cases. In all of these cases, the biopsies only harboured very superficially captured carcinoma. In only one case, downgrading was evident. Review of both specimens revealed that this case was a T1 HNSCC, in which a large incisional biopsy comprised the major part of the carcinoma leaving only residual invasive cancer within the resection specimen. Regarding the concordance of the two parameters budding and cell nest size in biopsy and the corresponding resection specimen, concordance rate of the morphologic pattern tumour budding was strikingly higher (κ = 0.87) compared with the pattern cell nest size (κ =  0.44). While tumour budding provides an overview of the dissociative growth capability, cell nest size represents the highest dissociative capability within the area of highest aggressiveness. This might be the reason why tumour heterogeneity plays a more prominent role in the evaluation of the cell nest size: biopsies by nature only capture a smaller part of carcinomas compared to resection specimen, potentially leading to a bias induced by tumour heterogeneity when evaluating cell nest size as the area of highest dissociative capability might not be included in the biopsy specimen. However, this bias particularly comes to effect in cases with scores 2/3 for cell nest size, and therewith obviously does not lead to a major effect on the sum score and the final grade, which is demonstrated by the high kappa value for the overall grading. Dik et al. analysed concordance of WHO-based grading of HNSCC between biopsy and resection specimens and found >50% discordantly graded cases in their studies.21 Concordance rates of histopathological grading in other entities as prostate cancer were higher with 50–70% concordant cases.29 The concordance rate of our grading is surprisingly high, which might be due to large and representative incisional biopsies performed at our institution.We found, that Tumour Budding and Cell Nest Size are of major prognostic relevance for patient outcome with presence of high budding activity and single-cell invasion indicating the most aggressive disease course with early relapses and poor survival. Summarising these two parameters and applying our CDG  discriminated three distinct prognostic groups. The prognostic value of the grading system was independent of potential additional influencing factors, such as age, gender and stage and revealed hazard ratios for OS  for nG2/nG3 HNSCC of 2.5 /4.2 . Again, previous studies revealed that the current conventional WHO-based grading of HNSCC does not show prognostic impact when applied in biopsy specimens.30 the “histological risk model” (HR)15 and the “tumour budding and depth of invasion” (BD)31 grading system. In contrast to our CDG, which has been successfully applied in oral SCC,25 in laryngeal and in hypopharyngeal SCC,24 and which has been transvalidated in oesophageal,26 cervical32 and pulmonary SCC,27 all of these approaches yielded varying results with respect to prognostic value and interobserver variability.37 Only the prognostic impact of the BD grading system, which evaluates BA together with tumour invasion depth, was confirmed in recent studies.31 However, per definition tumour invasion depth is a parameter that rather describes tumour stage than tumour grade as defined by the current TNM manual.39 Furthermore, this parameter is not applicable in biopsies, limiting its value for routine biopsy diagnostics.40Besides our grading approach, several alternative HNSCC grading systems have been proposed, including the “malignancy grading of invasive margins” (MG),42) but also in other solid tumours, such as colorectal44 and pulmonary carcinomas.27 Confirming our results, Tumour Budding has been shown to be of prognostic value not only in resection but as well in biopsy specimen of these tumour entities (reviewed in ref. 45). Focusing on the biologic background of Tumour budding, recent research has shown, that this histologic pattern might be the morphologic representative of epithelial-mesenchymal transition (reviewed in ref. 46). It has been identified as a sign of invasion and metastasis and has therefore been proposed as a novel hallmark of cancer by Makitie et al. (reviewed in ref. 47).Incorporation of markers of cellular dissociative capability such as Tumour Budding and Cell Nest Size into a grading system seems reasonable as presence of Tumour Budding has been shown to be associated with an unfavourable disease course not only in HNSCC  according to the CDG compared with well-differentiated carcinomas. In contrast, only Cell Nest Size but not Tumour Budding or CDG was correlated with pre-operative staging parameters (cT/cN). This might be due to the high discordance rate of pre-operative clinical cT and cN stage compared with post-operative pT and pN in 36%, respectively, 32% cases—a finding which confirms previous studies in HNSCC studies showing comparably low concordance rates between clinical and pathological staging.Our study clearly has some limitations as the included patient cohort contained a majority of advanced HNSCC. Furthermore, the study cohort included cases from different areas of the head and neck region . The study design was based on a prospective-retrospective concept with prospective sample collection from the day of the biopsy/resection followed by retrospective histopathological evaluation. Therefore, additional especially prospective study concepts and multi-centre studies are necessary to validate the prognostic impact of CDG in the pre-operative setting of HNSCC.25 we believe, that grading of HNSCC by CDG shows a promising potential for applicability in daily routine pathological and clinical practice. Taking together our previous studies,25 the major prognostic impact of the CDG was shown in the pre- and post-operative setting in the same homogeneous patient cohort as was the high inter- and intra-observer reproducibility of the grading system. Using the same patient cohort may prove the validity and significance of the results, as it allows to exclude a coincidental effect which might be observed when using different study cohorts.Taken together, our study revealed that CDG based on Tumour Budding and Cell Nest Size can successfully be applied in pre-therapeutic biopsies of HNSCC. The high prognostic impact of CDG assessed in biopsy specimen, separating three distinct prognostic groups, was independent of other prognostic parameters. Pre-operative grading was furthermore predictive for the presence of occult nodal metastasis, which had not been detected by routine clinical staging procedures in clinically nodal-negative necks. Considering the prognostic impact in the post-therapeutic setting and the high inter- and intra-observer reliability of the CDG which we demonstrated in previous studies,In conclusion, we believe that our findings provide a rationale for the integration of CDG assessment in routine diagnostics of HNSCC biopsies, since CDG has the potential to play a substantial role in treatment planning and prognostic patient stratification in HNSCC in the pre-therapeutic setting.Supplementary Material


Much interest has been expended lately in characterizing the association between DExH-Box helicase 9 (DHX9) dysregulation and malignant development, however, the enigmatic nature of DHX9 has caused conflict as to whether it regularly functions as an oncogene or tumor suppressor. The impact of DHX9 on malignancy appears to be cell-type specific, dependent upon the availability of binding partners and activation of inter-connected signaling pathways. Realization of DHX9’s pivotal role in the development of several hallmarks of cancer has boosted the enzyme's potential as a cancer biomarker and therapeutic target, opening up novel avenues for exploring DHX9 in precision medicine applications. Our review discusses the ascribed functions of DHX9 in cancer, explores its enigmatic nature and potential as an antineoplastic target. DExH-Box helicase 9 (DHX9) is an enzyme with multiple important functions in cells, therefore, its deregulated activity can cause alterations in cellular growth and subsequent formation of tumors. In particular, irregular DHX9 activity is attributed to the development of several hallmarks of cancer, however, it can have both pro- and anti-cancer effects, thus leading to conflicting views on DHX9's role in cancer development. The diverse implications of DHX9 in malignancy exposes the protein as a potential marker for cancer detection and intervention. Our review discusses the vast array of DHX9’s functions in cancer development, and its potential as a target for cancer treatment. Helicase proteins have a diverse range of cellular functions that stem from their ability to resolve and remodel DNA and RNA structures. DExH-Box helicase 9 (DHX9), also known as RNA helicase A (RHA), belongs to the DExD/H-Box superfamily II of helicases . The proThe unwinding and remodeling of DNA/RNA structures is ATP dependent and involves the catalytic action of helicases . DExD/H-DHX9 gene, a DExH-box helicase family member, maps to chromosome 1q25 in humans, a location that has been identified as the major prostate cancer (PCa) susceptibility locus . I. IEGFR ounctions . In thisctivator C 36]. p. pEGFR oortality . This hiferation .DHX9 is also capable of suppressing activity of the tumor suppressor BRCA1, which functions in the DNA damage response and cell cycle arrest ,78. BRCADHX9 inhibition in p53-deficient mouse lymphomas and colon cancer cells, highlighting the dependency of both p53-wildtype and p53-deficient tumors on DHX9 for survival [KIF1Bβ is often deleted in neuroblastoma tumors, thus impairing nuclear DHX9 localization and subsequently allowing neuroblastoma cells to evade apoptosis [p53 is a tumor suppressor that has been named ‘the guardian of the genome’ as it promotes cell cycle arrest or induction of apoptosis upon detection of DNA damage via enhanced production of proapoptotic proteins. p53 is commonly mutated or deficient in cancer, therefore allowing damaged cells to evade apoptosis, accumulate and promote cancer progression . As prevsurvival ,84. Convpoptosis . This woXIAP transcription via interaction with FUS, with both circ0005276 and XIAP overexpressed in PCa cells and tissues [XIAP expression, and subsequent PCa invasion and migration.One method by which DHX9 activity has been shown to influence cancer cell invasion is via interactions with lncRNAs leading to modulation of their activity. Lnc-cervical cancer DHX9 suppressive transcript (lnc-CCDST) binds DHX9, acts as a scaffold to enhance MDM2-DHX9 binding and thus induces DHX9 degradation. In cervical cancer (CC) cells, lnc-CCDST is downregulated via the human papillomavirus-encoded E6 and E7 oncoproteins, allowing DHX9 to accumulate and result in enhanced invasion and angiogenesis . DHX9 is tissues . Interes tissues , which pDHX9 knockdown in these cells induced STAT3 phosphorylation and subsequent proliferation, migration and invasion in vitro [Epithelial–mesenchymal transition (EMT) is a critical cellular process occurring during development that is characterized by a shift from epithelial to mesenchymal phenotype, however, cancer cells can hijack this event to promote transition to the more invasive mesenchymal phenotype, permitting escape from the primary tumor, invasion into the bloodstream and metastasis to distant locations . Many diin vitro . Upregulin vitro , therefoin vitro .MDR1 expression, and subsequently drives multidrug resistance in leukemia cells [SLFN11 expression compromises the DHX9-BRCA1 interaction, altering the regular DNA damage repair pathway and promoting chemoresistance [While the literature franking the involvement of DHX9 in the development and progression of cancer is robust and plentiful, a newly discovered feature of DHX9 revolves around resistance to chemotherapeutic agents. The majority of chemotherapy drugs, such as platinum-derived agents, target highly proliferative cells and induce DNA damage that triggers cellular DNA damage responses and repair pathways, however, upon irreversible DNA damage cell death is programmed via apoptosis . Resistaia cells . Additioia cells . Contraria cells . Given tia cells , dysregusistance .DHX9 induces activation of the p53 signaling pathway in a mouse lymphoma model. In this context, the activated replicative stress response induced apoptosis and sensitized resistant lymphoma cells to ABT-737 [DHX9 would usually target it to nonsense-mediated mRNA decay, however, in Ewing sarcoma DHX9 expression is upregulated following repression of this exon and correlates with a poor prognosis [DHX9 exon 6A to suppress its inclusion and promote DHX9 expression. Downregulation of these proteins inhibits DHX9 expression, suppresses proliferation and sensitizes Ewing sarcoma cells to doxorubicin treatment, revealing the importance of DHX9 alternative splicing in conferring chemosensitivity or chemoresistance [Analysis of a cadre of RNA/DNA hybrid-binding proteins has revealed that DHX9 has one of the highest levels of drug sensitivity interactions with the US FDA-approved chemotherapeutic agents, exhibiting both positive and negative correlations, highlighting the fact that DHX9 expression can variably affect chemotherapy response . p53-ind ABT-737 . Concept ABT-737 . Inclusirognosis . A diffesistance .et al. revealed that DHX9 depletion in mice induced embryonic lethality, thus proving essential for embryonic development [DHX9 knockdown was lethal to a number of cancer cell lines and lymphoma cells of a mouse model while imposing no detrimental effects on healthy tissue in vivo. Additionally, adult mice seemed to tolerate long-term genetic DHX9 suppression. Of particular importance in this regard is the fact that in vivoDHX9 knockdown did not negatively affect other highly proliferative cells, ensuring high specificity while exhibiting low toxicity to healthy tissue [DHX9 suppression in p53-null tumors also induced cell death or cell cycle arrest [in vivo. The aptamer binds DHX9 with high affinity and specificity allowing preferential action in cancer cells over normal tissue due to DHX9 overexpression in CRC cells. Further specificity is achieved by localizing the aptamer to the nucleus allowing targeted delivery and this approach did not negatively affect normal cells. Given that this work was performed in human xenografts, it is highly likely that such a method could translate to humans, further establishing DHX9 as an effective and safe molecular target [One of the main pitfalls of chemotherapy is lack of specificity due to the general targeting of highly proliferative cells as opposed to only bona fide cancer cells, which can result in highly toxic cellular conditions that inadvertently damage healthy tissue . Targeteelopment , severaly tissue . A folloe arrest . Given te arrest , this var target . Anotherr target . FurtherCancer immunotherapy can offer more selective and tailored treatment than chemotherapy by hijacking the immune system to recognize and destroy cancer cells . JP Alliet al. identified that DHX9, along with several other DExD/H-box helicases, provokes antiviral responses by attenuating MYXV viral replication in human cancer cells. siRNA-mediated DHX9 knockdown enhanced MYXV replication in cancer cells, showing that DHX9’s regulatory role in the antiviral immune response is highly relevant in this context [DHX9’s aforementioned roles in innate immunity with respect to antiviral immune responses and viral replication places the helicase in a pivotal position to hone cancer cells’ responses to oncolytic viral therapies. Myxoma virus (MYXV) is a potential candidate for oncolytic viral therapy because of its ability to infect human cancer cells both efficiently and safely ,117. Rah context . However context . Interes context , therefo context . It was  context . OverallIt is clear from the literature cited above that DHX9 has well-defined roles as an oncogene and a tumor suppressor in different tissues and cancer cell types. The versatility of the helicase is a product of its multiple functions in resolving nucleotide structures and also of the numerous protein–protein interactions it makes with binding partners that transduce vital signals for fundamental cellular processes such as gene expression and DNA replication. DHX9’s multitude of functions in the development of numerous hallmarks of cancer highlight a pivotal role in malignancy and potential as both a biomarker and selective target for cancer therapy.There is growing evidence to suggest that DHX9 would make an effective therapeutic target and possibly a diagnostic gene for some cancers, however, careful thought is needed to hone approaches that selectively focus on the oncogenic potential of the helicase rather than the plethora of vital ‘housekeeping’ functions it undertakes to maintain the stability of the human genome. The proposal of DHX9 as a potential target for enhancing oncolytic viral therapy is an avenue to be further explored and given the recent advances in cancer immunotherapy, it can be assumed that this field will continue to evolve rapidly. Ultimately, targeting DHX9 for inhibition appears to offer a safe and selective approach for cancer treatment, and combined with current cancer therapies could lead to more effective and specific treatments applicable to a broad range of cancers, however, clinical trials are lacking in this area, therefore further research is required to fully ascertain its full potential.The hallmarks of cancer are features of cancer cells that promote/support the development and progression of cancer.DExH-Box helicase 9 (DHX9)’s enigmatic functions include maintenance of genomic stability, DNA replication and gene expression. Dysregulated DHX9 activity leads to a lack of coordination of these important cellular processes, which in turn can lead to the development of numerous cancer hallmarks and malignancy.DHX9 can function as either an oncogene or tumor suppressor dependent on its interaction with downstream partners and activation state of inter-connected signaling pathways.The involvement of DHX9 in malignancy positions it as an attractive biomarker and target for cancer therapy.


We subsequently tested 200 patients using the OrientGene COVID-19 IgG/IgM Rapid Test Cassette and find a sensitivity of 74% in the early infection period (day 5–9 post symptom onset), with 100% sensitivity not seen until day 13, demonstrating inferiority to PCR testing in the infectious period. Negative rate was 96%, but in validating the serological tests uncovered potential false-negatives from PCR testing late-presenting cases. A positive predictive value (PPV) of 37% in the general population precludes any use for general screening. Where a case definition is applied however, the PPV is substantially improved (95.4%), supporting use of serology testing in carefully targeted, high-risk populations. Larger studies in specific patient cohorts, including those with mild infection are urgently required to inform on the applicability of POC serological assays to help control the spread of SARS-CoV-2 and improve case finding of patients that may experience late complications.Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic. Case identification is currently made by real-time polymerase chain reaction (PCR) during the acute phase and largely restricted to healthcare laboratories. Serological assays are emerging but independent validation is urgently required to assess their utility. We evaluated five different point-of-care (POC) SARS-CoV-2 antibody test kits against PCR, finding concordance across the assays ( SARS-CoV-2 has now extensively spread and been designated a global pandemic.On 31 December 2019 alarm over a pneumonia-like viral illness caused by an unknown pathogen was first raised to the World Health Organisation. A genome sequence for the presumed causative agent since designated Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) was publically released by early January 20202. The burden on healthcare systems has been devastating, particularly for intensive care services3. As of April 2020 there had been over 1 million cases with a case fatality ratio varying from 1 to 6%4. The true case fatality ratio is thought to be closer to 1–2% with a considerable number of unrecognised subclinical cases likely to be found in the community. Without the availability of serological testing, this has proven very difficult to accurately assess2. While real-time PCR is available during the acute infection it is limited by laboratory and skilled operator requirements5, and can only be used early in the symptomatic clinical course. Serologic testing for SARS-CoV-2 offers the potential for enhanced case finding for patients presenting later in the disease course, as well as enabling serosurveillance at a much wider level than molecular diagnostics allow.Clinical presentation ranges from mild upper respiratory tract infection to severe pneumonia with acute respiratory distress syndrome6 against the current gold standard of RT-PCR. We go on to take one assay forward for further evaluation (OrientGene COVID-19 IgG/IgM Rapid Test Cassettes), completing 200 assays and commenting on test performance metrics in a UK inpatient population.Multiple lateral flow serology tests have recently been developed for rapid, point-of-care (POC) use, providing qualitative results within 15 min. Whilst many of these devices have their initial development in China, they require verification in the cohort intended for use before extensive uptake. We take five of these POC kits and evaluate their suitability and potential utility for use in a western European population that had experienced symptoms in line with the Public Health England (PHE) case definition for testingFifteen patients  underwent testing with each of the five assays. All patients had RT-PCR testing conducted between 2 and 10 days from symptom onset, except patient 11 who was first tested at day 13 . The same seven patients were reactive across all five serology tests  were tested with each of the five assays . The same four patients were unreactive across all five serology tests (20/25). The fifth patient was reactive using all five assays (5/25).200 patients  underwent testing. All 200 assays were included in the final analysis. Two test kit failures were observed (failure of solution to ascend chromotography paper), of which both were repeated with subsequent valid reads.n = 50) 37/50 were reactive . At days 10–14 post-symptom onset (n = 50), 43/50 were reactive . At greater than 14 days post-symptom onset (n = 50) there were 50/50 reactive assays  (Table n = 67)  were included . The tests with too little blood (n = 1), or too much blood (n = 1) failed, with no reactivity seen on test lines in the former and no reactivity in test or control lines in the latter.Re-testing of patients with reactive assays with a second test had demonstrable reproducibility  99.6%. When calculated for suspected cases meeting the PHE criteria using a prevalence of 48% (86/177 positive in those tested across the week of 23 March–29 March 2020) in our inpatient population at the same time point there is a PPV of 95.4% and NPV of 88.4%.Where an estimated prevalence of 2.7% in the general population is applied the positive predictive value (PPV) is calculated at 37%6 during this early validation study makes it difficult to draw high-powered conclusions, the results offer useful insights into the potential utility of POC serological testing at this early stage in the pandemic response. Key concerns exist around the reliability of serology testing and identifying any potential time points for appropriate use.Serologic testing for SARS-CoV-2 has the potential to augment control strategies by improving our understanding of the true burden of infection during the current pandemic. While the small number of tests performed 7. Practically, if considering potential use of the assays in the early phase of infection  the sensitivity drops to 66% (n = 32). It is not until the end of the second week from symptom onset that the test appears to demonstrate a more practical use. In the greater than 14 day since symptom onset group there was 100% sensitivity. The latest false negative was demonstrated on day 12 with reactive results produced in all tests taken thereafter (n = 67) suggesting that there could be real utility in POC serology from 14 days onwards. Where PCR testing is currently limited due to demand this could therefore have a potential role in testing those healthcare workers who have already self-isolated because of possible infection in the last few months. In addition the assay may have a role in community testing where timelines in patients with mild symptoms already self-isolating would be less time critical. A role is not seen however for an impact on public health guidance around self-isolation practices during the infectious period. A PPV of 37% for the general population precludes any consideration of deployment of serology testing for general screening. Where a case definition is applied however, as is required for meeting the PCR testing criteria, the PPV is substantially improved (95.4%) and supports use of serology testing in carefully targeted populations. While limited to detection in targeted populations this would still enable a far better understanding of the infection-fatality ratio then we are currently able to draw from predominantly hospital cases of infection.Our study raises significant concern for the reliance on serology testing in the initial infection phase. Analysis of the larger 200 patient cohort shows that when the initial patient group (days 5–9 post symptom onset) are considered, there is a sensitivity of 74%, with the potential therefore to miss a significant number of positive cases during their most infectious period7. If the ‘negative’ patients across both halves of the study  are considered, three had reactive results, with only one demonstrating a reactive IgG line. Given our selection of this ‘negative’ patient from among those who had an a priori clinical suspicion of SARS-CoV-2 infection but who were PCR negative, it is possible or indeed likely that these represented missed cases of SARS-CoV-2. This particular patient presented in extremis with acute respiratory distress-like features  demonstrated a negative rate of 96% suggesting an additional role in supporting suspected inpatients that return negative PCR results. In particular this could help guide management of patients presenting outside the reliable detection period for PCR swabbing of the upper respiratory tractWhen considering use in outpatient settings, it is vital that a suitable time period is established for a true negative result. Our study suggests that this point is to be found at day 13 post-symptom onset at the earliest, limiting the utility of these assays in enabling return to work or in informing additional self-isolation practices. There is however a real role in helping to identify those healthcare workers and so better understand overall the overall burden of infection in high-risk groups, that have been through a self-isolation period with presumptive infection supported by concomitant reinforcement of advice around the stringent infection, prevention control measures.If widely employed, demand on suppliers for POC serological assays will be high and interchangeability of kits may therefore be required. The plurality of kits currently available makes it difficult to comment on individual assay performance at this early stage and it is therefore essential to evaluate performance across the range of kits available both against the gold standard (PCR) as well as against each other as soon as possible. Of the five different assays we evaluated, there was direct concordance for detection of SARS-CoV-2 infection across seven of the PCR positive patients (35/38 of reactive results). All five kits failed to detect SARS-CoV-2 infection from two known early positive samples taken at day 5 and day 6 post-symptom onset. Our findings suggested that results consistently appeared reactive from only day 8 post-onset of symptoms onwards across all five kits . We cite ongoing concern for their reliability during that early period and the significant impact that missing a positive infection could have on public health control measures.8.All POC serology kits are qualitative and while there was general correlation with colour intensity as time from symptom onset increased, variance in intensity of positive results was noted. The test kit instructions comment clearly on expectation for varied colour intensity of positive results and advise that even very weak colour should be considered to be reactive. When considering the practicalities of widespread testing, clear instruction on interpretation will have to feature in standard operating procedures and should allow for comment on interpreting the colour intensity of reactive samples. Antibody levels analysed via validated laboratory-based enzyme-linked immunosorbent assay (ELISA) platforms have suggested higher levels following severe compared to mild infections and cohort studies will now be needed to evaluate the extent of reliability for POC serology testing in mild community infectionLittle in the way of conclusion can be drawn from the five test kits used to explore concerns around end-user difficulties. Re-sampling of reactive patients provided re-producible results and further investigation should be conducted to comment on reproducibility of POC serology kits. Of significant use however is the observation that tests completed against manufacturer protocol by transferring either too little, or too much whole blood into the well resulted in failure. Those tasked with carrying out testing must complete the test as instructed using the marked pipette in order to have reliable results. Failure to register a control line in these cases should avoid the mistake of reporting an inappropriate false result.n = 130), 119 showed both IgM and IgG reactivity. There were 11 reactive samples that differentiated between IgM (6/130) and IgG (5/130)  versus the SARS-CoV-2 total antibody test (Wantai), the former have a potential capacity to provide greater information. Of the total reactive tests in the 200 cohort study n = 30, 119 s. used serum samples from 3 SARS-CoV-2 positive patients and found cross-reactivity with the SARS-1 S and S1 proteins, and less so but also with MERS-CoV S protein8. The S1 protein appears to be more specific to the SARS coronaviruses, with cross reactivity not seen against an array of PCR positive samples for example seasonal human coronavirus OC43 as well as non-CoV pathogens, including EBV and CMV. Subsequent testing with IgA and IgG specific ELISA showed some cross-reactivity with OC43 PCR positive samples and highlights the importance of assay design, where perhaps multiple protein targets may be required to ensure specificity for SARS-CoV-28. This urgently needs investigation in the western European population before widespread role out of POC SARS-CoV-2 serology. The role of serology as an adjunct to PCR has also recently been reported, with a strong correlation seen between IgM and IgG and the severity of disease10. Seroconversion for detection of IgM and IgG was seen at median day 12 and 14 respectively with possibility of detection much earlier in the clinical course and is supportive therefore of our findings of improving sensitivity through the first 14 days of infection10.Cross-reactivity leading to false positive results is of considerable concern, both with human seasonal coronaviruses and zoonotic beta-coronaviruses, including SARS and MERS. Okba et al11.If shown to be successful in the community setting POC serology testing could have significant implications for tackling the pandemic in resource poor settings which lack the required infrastructure for centralised molecular techniques. The benefits of utilising point-of-care testing to improve the efficiency of case identification and resource allocation in such circumstances has previously been discussed during the Ebola epidemic and could therefore be of considerable use during the SARS-CoV-2 pandemic12. While our cohort groups include immunocompromised patients, pregnant individuals, and elderly patients, we are unable to sufficiently comment on reliability in these groups. This study is limited to evaluating reactivity in symptomatic adults with moderate-severe disease only and response in children has not been investigated. All positive patients in this study were considered to have moderate-severe infection by nature of the admitting policy of the hospital and evaluation in those with mild disease symptoms is needed to comment further on the population level applicability of these assays.The small number of assays evaluated in this study limits the degree to which conclusions can be drawn. Formal power calculations have not been conducted, but with a sensitivity of 100% at greater than 14 days, accompanied by a margin of error of only 0.04, this exploratory study could now inform larger scale appropriately powered studies to fully characterise the utility of these assays in specific population groups. A greater number of samples are now required in order to comment reliably on sensitivity and specificity and in turn, once the evolving prevalence becomes clearer, the positive and negative predictive value of these tests in various groups. It would also be useful to conduct specificity testing using pre-pandemic serum in order to better assess possible cross-reactivity. The need for rapid initial verification of these assays to augment clinical and public health interventions in this pandemic does not yet enable comment on many aspects of the assays, including the IgM/IgG relationship in the weeks and months post-infection. Awaited longitudinal studies will allow evaluation of assay performance at extended points in the convalescent period and better define an appropriate testing window. Additionally, where results are qualitative and vary in strength of response, it is unclear how test sensitivity would differ when (i) healthcare professionals not used to evaluating lateral flow devices or (ii) the general public read results. Subsequent work has since provided a model for reading SARS-CoV-2 serological assays however which may help with reduce any loss of sensitivity from reading by those less experiencedSARS-CoV-2 has resulted in a global pandemic and is associated with considerable morbidity and mortality. Serology testing is easy-to-use at the point-of-care and its utility to support, and replace where appropriate, PCR could allow for a significant improvement in the understanding of the true infection burden. This will be vital to the control of further SARS-CoV-2 spread.Serology testing appears to be unreliable during the early infectious period, however there may be a limited role for use of delayed-case identification in the appropriate context, such as in any patient that is at least 14 days post-symptom onset. Our data would suggest an inferiority to PCR testing in the acute phase of infection, making them inappropriate for use in diagnosis during the infectious period. Further evaluation on a much larger scale is now necessary in order to reliably comment on the earliest stage post symptom onset at which testing can be utilised, as well as on its performance in different population groups. In the meantime, any use of rapid serological test kits must be interpreted with caution, particularly if symptoms are of 14 days or less duration.13, and had an accurate history recorded of at least 5 days of symptoms.A prospective single-centre study was completed at a 430-bedded acute London hospital during March 2020. Consecutive inpatients were selected for testing based on real-time PCR detection of SARS-CoV-2  and stratified based on duration of symptoms. Patients were included if they were admitted to hospital with symptoms matching the PHE case definition for testingExclusion criteria included all patients under 16 years old. Mild infection not requiring admittance to hospital were excluded. Patients that were unable to provide an accurate history or those with vague onset of symptom duration were also excluded in order to accurately report time-lines.15 SARS-CoV-2 CE marked split IgM/IgG antibody tests (Core tests) were available for paired testing alongside 15 SARS-CoV-2 CE marked total antibody rapid tests (Wantai), 15 COVID-19 CE marked split IgM/IgG Rapid Tests (VivaDiag), 15 COVID-19 CE marked split IgG/IgM Rapid Test Cassettes (OrientGene), and 15 SARS-CoV-2 CE marked split IgM/IgG One Step Rapid Test Devices (Encode). Ten PCR positive and five PCR negative patients were selected and paired serology testing was carried out by the same three doctors  experienced in the use of rapid diagnostic test kits. In order to evaluate reliability of test reactivity in positive patients, five positive patients were evaluated between days 5–9 of symptom duration and five patients between days 10–14. Additionally, five patients thought to be true negative ((i) symptoms meeting the case definition lasting at least 5 days but no more than 10 days at presentation, (ii) at least one negative SARS-CoV-2 PCR result, and (iii) an alternative clinical diagnosis for their symptoms) underwent paired testing in order to assess for false positive reactivity.Each patient was tested by the same doctors, as per each product protocol. Test kits were then read at the designated time period as per individual protocol and a result recorded for both the appearance of a control line and the presence or absence of antibody test reactivity. All results were verified independently by two of the doctors.A power-calculation for assessing significance of sensitivity analysis (with an estimate of 90%) was applied in analysis, setting type I error at 0.05 and type II error at 0.20. Assuming assays performed as per the manufacturer information it was calculated that testing 132 PCR positive individuals would be required. Using the same inclusion criteria a further 200 patients were evaluated using the COVID-19 IgG/IgM Rapid Test Cassettes (OrientGene). Patient history from both the Emergency Department assessment and the admission clerking were carefully evaluated and patients excluded if there was doubt over exact symptom onset, including those patients with vague history of onset, presenting with confusion, or following early intubation with an incomplete history. Patients were stratified based on consecutive PCR positive results to one of 3 time interval groups since symptom onset, encompassing 83% of SARS-CoV-2 positive cumulative inpatients at the time of the study period end-point. 50 PCR positive patients were stratified to the 5–9 post-symptom onset group, 50 patients to the 10–14 day post-symptom onset group and 50 patients at greater than 14 days since symptom onset. An additional 50 patients were selected with a presumed negative diagnosis with a negative SARS-CoV-2 PCR result, admitted with an acute surgical complication or had a fever with an alternative, non-respiratory primary diagnosis.6, were used to estimate a PPV if these assays were to be deployed for use in the general population.Prevalence estimates at the time of the study, provided by modelling by the MRC Centre of Global Infectious Diseases Analysis reportFive additional test cassettes were used to evaluate possible anticipated end-user difficulties. Three test cassettes were employed to retest a patient with a positive result to comment on reproducibility. One test cassette was completed with too little blood, failing to fill the pipette to the indicated line and one test cassette was completed with too much blood, by over-filling the well.This study was approved by the Chelsea & Westminster NHS Foundation Trust COVID testing committee. The study was reviewed by the Chelsea & Westminster NHS Foundation Trust Research and Development Office and deemed a verification of a CE marked in vitro diagnostic test, therefore the need individual informed consent was waived. Aggregated data was analysed in accordance to the UK Secretary of State for Health and Social Care general notice that under the Health Service Control of Patient Information Regulations (2002) patient data for a COVID-19 purposes may be used for research. The study was conducted in accordance with relevant guidelines and regulations including the Declaration of Helsinki.


Primary coenzyme Q10 deficiency is a rare disease that results in diverse and variable clinical manifestations. Nephropathy, myopathy and neurologic involvement are commonly associated, however retinopathy has also been observed with certain pathogenic variants of genes in the coenzyme Q biosynthesis pathway. In this report, we describe a novel presentation of the disease that includes nephropathy and retinopathy without neurological involvement, and which is the result of a compound heterozygous state arising from the inheritance of two recessive potentially pathogenic variants, previously not described.Retrospective report, with complete ophthalmic examination, multimodal imaging, electroretinography, and whole exome sequencing performed on a family with three affected siblings.COQ2 gene, resulting in a frameshift variant in one allele, and a predicted deleterious missense variant in the second allele  and c.376C > G,p.(Arg126Gly) respectively). Electroretinography results were consistent with rod-cone dystrophy in the affected individuals. All affected individuals in the family exhibited the characteristic retinopathy as well as end-stage nephropathy, without evidence of any neurological involvement.We show that affected individuals in the described family inherited two heterozygous variants of the COQ2 gene that result in primary coenzyme Q deficiency. Targeted sequencing of coenzyme Q biosynthetic pathway genes may be useful in diagnosing oculorenal clinical presentations syndromes not explained by more well known syndromes .We identified two novel compound heterozygous variants of the  Endogenous defects in the biosynthesis pathway of coenzyme Q result in mitochondrial respiratory chain deficiency and associated manifestations. Involvement of the disease is systemic, and often includes neurologic manifestations , kidney manifestations , hypertrophic cardiomyopathy, myopathy and sensorineural hearing loss [COQ2 and PDSS2 have been associated with retinopathy and optic atrophy, respectively. Depending on the severity of the variant and the deficiency, manifestations may be severe and appear in infancy or mild and begin in adulthood.Coenzyme Q10  were conducted. Patient consent was obtained prior to genetic testing. All information in this report has been de-identified in accordance with HIPAA and institutional review board regulations.After measuring best corrected visual acuity, all patients’ eyes were dilated using topic 1% tropicamide and 2.5% phenylephrine followed by complete ophthalmic assessment including funduscopic examination, digital fundus photography, fundus short-wavelength autofluorescence (SW-AF) and spectral domain optical coherence tomography (SD-OCT). SW-AF  and SD-OCT images were taken by the use of a confocal scanning laser ophthalmoscope . SD-OCT images were acquired as horizontal 9 × 9 mm scans  positioned through the macula and obtained in high-resolution mode. The scans were recorded automatically by the use of recorded IR-R (820 nm light source) fundus images.Full-field electroretinogram (ffERG)  was performed with Dawson Trick Litzkow (DTL) fiber electrodes and Ganzfield stimulation. ERG recordings were obtained for our patient for both eyes in accordance with the International Society for Clinical Electrophysiology of Vision (ISCEV) guidelines in both the scotopic and photopic states . The ampES was performed on the three affected siblings at the Laboratory of Personalized Genomic Medicine (PGM) at Columbia University Irving Medical Center (CUIMC) on DNA obtained from peripheral blood. Written consent was obtained, exome sequencing libraries were prepared from genomic DNA from the probands using Agilent SureSelectXT (Human All Exon v.5 + UTRs) capture kit according to the manufacturers’ protocol. Paired-end sequencing was performed on the Illumina HiSeq 2500 platform. The sequence data were aligned and annotated using NextGENe  software. Variant filtering and annotation were performed using an in-house developed proprietary analytical pipeline. Variants of interest were further analyzed using VarSome [A 25-year old man of Ashkenazi Jewish (AJ) descent presented to the clinic with new-onset difficulty with night vision. His medical history was notable for biopsy-proven diffuse glomerulosclerosis and associated hypertension. Immunofluorescence studies of his renal biopsy demonstrated no evidence supporting immune complex type disease, and the majority of glomeruli examined were globally sclerotic. Best-corrected visual acuity was 20/30 in the right eye (OD) and 20/25 in the left eye (OS), with no afferent pupillary defects. Visual fields to confrontation were constricted; anterior segment exam was unremarkable. His dilated fundus exam revealed healthy-appearing nerves and a flat normal-appearing macula, and clear vitreous in both eyes. Notably, he had extensive intraretinal pigment migration in the periphery arranged in a ring-like fashion, encircling the posterior poles in both eyes Fig. a. AutoflThe patient’s family history was notable for two of four other siblings with similar ophthalmic and medical histories. The parents showed no manifestation of disease and denied familial consanguinity. His first affected sibling was an older brother of 32 years of age, who was diagnosed with “retinitis pigmentosa” at age 21, and who had received a kidney transplant at age 5 years for a diagnosis of mesangial sclerosis. His medical history was also notable for elevated liver enzymes of unknown etiology and childhood acute lymphoblastic lymphoma that went into remission. On ophthalmologic exam, this sibling demonstrated a best corrected visual acuity of 20/25 OD, 20/30 OS, absence of relative afferent pupillary defects, and constricted visual fields to confrontation. Corneas were clear and the anterior segments were quiet, but he had a P1 posterior subcapsular cataract  in both The patient’s second affected sibling was a 28-year old sister, also with a history of “retinitis pigmentosa” diagnosed at age 23, and a kidney transplant at age 10 for end-stage renal disease of unknown etiology; her birth history was notable for an extra digit on the right foot. Similar to her two affected siblings, her best-corrected central visual acuity was relatively good at 20/30 in both eyes (OU), without relative afferent pupillary defects. Her visual fields to confrontation were constricted, her corneas were clear and her anterior segments were quiet. Her lenses contained a P1 posterior subcapsular cataract  in both Electroretinography (ERG) was performed on all three siblings and revealed a pattern consistent with rod-cone dystrophy, with an undetectable scotopic response Fig. . All affCOQ2 (NM_015697.8) that segregated in all affected individuals  and Bardet-Biedl syndrome 1–21 , no variants were detected in anyone of the corresponding genes; ES .This variant was detected at an allele frequency of 6.987e-3 in gnomAD v3 exomes and genomes, seen in the heterozygous state in 10/143116 alleles, with no homozygotes. This suggests that it is not a common benign variant in the populations represented in these databases. A literature search revealed no previous patient reports described with this variant. There are conflicting predictions of pathogenicity for this variant in ClinVar (VarID: 631951), partly due to lack of reports in patients and higher allele frequency in the gnomAD Ashkenazi Jewish population . Though pathogenic frameshift variants reported in this gene are rare, there is one frameshift variant p.(Leu234Argfs*14) that terminates the protein downstream of this variant reported in the literature in a patient with isolated nephrotic syndrome  substitutes a glycine for an arginine at a conserved amino acid residue 126/422 in the hydroxybenzoate polyprenyl transferase domain of COQ2 :c.288dupC,p.(Ala97Argfs*56) and COQ2(NM_015697.8):c.376C > G,p.(Arg126Gly), which together in the compound heterozygous state led to primary CoQ10 deficiency in three siblings. Due to limitations imposed by the COVID-19 pandemic, we were unable to confirm our genetic findings through measurement of CoQ10 levels from muscle biopsy or fibroblast samples. However, the pathogenicity of these variants is supported by several observations, including manifestation of symptoms in all three affected family members, population database analysis and in silico predictions. The combination of these variants in the compound heterozygous state primarily presented as severe progressive glomerulosclerosis leading to eventual kidney transplant at a young age, as well as progressive retinal degeneration mimicking retinitis pigmentosa.Here we describe two novel recessive potentially pathogenic variants: COQ2 gene and clinical manifestations, including neurologic and renal involvement, have previously been reported in the literature :c.890A > G,p.(Tyr297Cys). The other two patients were siblings with a homozygous variant, COQ2(NM_015697.8): c.328A > G, p. that manifested as multiple-system atrophy and retinitis pigmentosa without clinical signs of nephropathy [The  altered . Althouginopathy . His affhropathy . One of hropathy . The othhropathy , and theCOQ2 is the only gene in the CoQ10 biosynthesis pathway that has been definitively linked with a retinitis pigmentosa-like presentation in the reported literature. There are two additional reports of patients with severe CoQ10 deficiency and retinopathy without confirmed variants in any of the biosynthetic pathway genes; rather, CoQ10 deficiency in these cases was confirmed biochemically. Severe neurological impairment at a young age was observed in both of these cases. In one of the reported cases of a young patient with severe coenzyme Q10 deficiency, retinopathy was demonstrated by an abnormal electroretinogram and mild pigmentary degeneration of the retina [COQ1 gene in this latter case revealed no causative variant. Other genes in the biosynthetic pathway have been associated with other ocular abnormalities other than pigmentary retinopathy, such as such as optic atrophy . Why COQ2 is the only gene thus far to definitively cause pigmentary retinopathy remains to be determined.Interestingly, e retina . In the e retina . SequencCOQ2 variants. The efficaciousness of CoQ10 may be higher in cases where supplementation is started at an earlier age and prior to the onset of irreversible damage to the retina. In previously reported patients with retinitis pigmentosa stemming from variants in the COQ2 gene, treatment with CoQ10 supplementation was not administered [COQ2 gene, after CoQ10 supplementation, neurologic manifestations improved significantly, but there were no noted effects on renal function [COQ2 variant exhibiting renal failure [Attempted treatment with high dose CoQ10 supplementation in this family failed to improve the ERG signal or vision, however vision remained stable, and there was no progression of retinopathy was observed on autofluorescence or in terms of retinal atrophy on optical coherence tomography. This may suggest that CoQ10 supplementation can aid in preservation of viable retina even if there is no improvement in areas that are already affected. Thus, CoQ10 supplementation in patients with retinopathy should be considered as a possible treatment to stabilize remaining retinal function in individuals with the ocular complications associated with pathogenic nistered , 27. In function , 19. In function . Respons failure . Twenty  failure .Variable response to supplementation by CoQ10 may also be due to variable bioavailability of CoQ10 or CoQ10 analog supplementation. Several studies have investigated the use of CoQ10 precursors that may sidestep the defects in the synthesis pathway, and such precursors may be another consideration for treatment in similar families to the study family described. One such precursor is 4-hydroxybenzoic acid (4-HBA), which is a substrate for COQ2. Although this molecule does not bypass the COQ2-mediated enzymatic step in the CoQ10 synthesis pathway, one study showed that supplementation of COQ2-deficient fibroblasts in vitro with 4-HB appeared to overcome the enzymatic defect in a dose-dependent manner, implying that certain defects may be overcome by saturating the defective COQ2 enzyme with this CoQ10 precursor . Other sCOQ2 gene, resulting in primary coenzyme Q10 deficiency that manifests primarily as retinitis pigmentosa and renal failure. Unlike other primary CoQ10 deficiency syndromes previously described, these patients do not display neurological dysfunction at presentation. We believe this rare entity should not be missed when considering the wide differential in patients with oculorenal syndromes, as it is potentially treatable if caught early.We present in this report a rare oculorenal syndrome in three related patients associated with novel compound heterozygous variants in the


The proximity required of a thorough biomicroscopic slit-lamp examination may put ophthalmologists at increased risk for respiratory-borne infection with SARS-CoV-2. Conjunctivitis has been described in a few patients with COVID-19 and other coronavirus syndromes. Although SARS-CoV-2 has been detected in the conjunctival secretions or tears of patients with COVID-19 and conjunctivitis, transmission of infection through respiratory droplets to ophthalmologists without eye protection or masks may be the bigger concern. Many systemic viral infections have ocular manifestations; examples include adenovirus,2 enterovirus 70, H1N1, SARS coronavirus (SARS-CoV),3 and human coronavirus NL63,4 the last of which was, in fact, first identified in a 7-month-old child with conjunctivitis and bronchiolitis.4 Therefore, it may not be surprising that ophthalmologist Dr Li wisely deduced that an incipient SARS-like viral outbreak was occurring in Wuhan, China, well in advance of others because coronavirus syndromes are known to be associated with conjunctivitis.Although generally considered a surgical subspecialty, ophthalmology involves much “chair time,” involving close contact with patients’ aerosolized respiratory droplets or tears and often manipulation of eyelids and conjunctiva during slit-lamp examination. Few (if any) ophthalmologists routinely wear respiratory masks or gloves unless the patient appears to have a respiratory infection or conjunctivitis with tearing or other discharge. Few are aware that the late Dr Li Wenliang, who alerted Chinese government officials of an incipient viral syndrome that would be named COVID-19, was a young ophthalmologist. By his account, he believed he contracted the infection from a glaucoma patient. It is unclear whether the patient had conjunctivitis5 and in the 2 eyes of a patient who went from home quarantine to hospital admission during which time this patient developed bilateral follicular conjunctivitis.6 Conjunctivitis was the initial symptom of a third individual who was part of a Chinese expert panel investigating the outbreak in Wuhan and developed COVID-19.7 The prevalence of “conjunctival congestion” was 0.8% in 1,099 cases of confirmed COVID-19, but 8 of these 12 patients had severe or critical disease requiring ventilation,8 which suggests that they may not have had conjunctivitis. In COVID-19 patients with conjunctivitis, SARS-CoV-2 has been recovered from conjunctival secretions, posing a potential route of ocular transmission.6 Patients who present to ophthalmologists with isolated conjunctivitis may already be infected with and secreting SARS-CoV-2 in tears and elsewhere, but infected persons can also be asymptomatic or presymptomatic. Thus, universal precautions that should be reflexive standard ophthalmic practice apply more than ever. However, the risk of transmission through tears or conjunctival secretion is probably small relative to respiratory transmission.Conjunctivitis can be a clinical manifestation of coronavirus 19 (COVID-19). It was described in the 2 eyes of the single patient with conjunctivitis in a series of 30 patients who had “common” and “severe” forms of COVID-19,With the COVID-19 outbreak, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a concern that ophthalmologists may be inherently at increased occupational risk for infection. The essential nature of encounters in ophthalmology clinics necessitates patient and ophthalmologist to be separated by <20 cm during slit-lamp biomicroscopic examination and <5 cm in situations requiring direct ophthalmoscopy, yet the World Health Organization recommended “1 meter (3 feet) distance between yourself and anyone who is coughing or sneezing” and the Centers for Disease Control and Prevention recommended 6 feet from anyone. “Physical distancing” is not an option for ophthalmologists.7 By at least one account, a member of a Chinese national expert panel on pneumonia reported that he was infected via ocular exposure during an inspection in Wuhan. He was wearing an N95 mask but no eye protection. Several days before he developed COVID-19, he developed bilateral redness of his eyes.7 In the 2003 Toronto SARS outbreak, 7 of 45 laboratory-confirmed SARS patients requiring intubation transmitted SARS-CoV to 26 of 697 healthcare workers.9 The highest estimated healthcare worker risk in generalized estimating equation models was eye or mucous membrane exposure to body fluids , while in the classification and regression trees analysis, the primary healthcare worker related risk factor was whether or not eye protection was worn.9 This analysis does not mean that conjunctival contact is a primary mode of spread of SARS-CoV but that the conjunctiva is a possible portal of entry for SARS-CoV and SARS-CoV-2 given the ocular tropism of other respiratory viruses like human coronavirus NL63 and adenovirus.Because of physical proximity, a presymptomatic patient with COVID-19 could transmit disease by breathing or coughing on an ophthalmologist. Another mode of transmission that is underappreciated is that infectious body fluids and aerosol droplets can be transmitted to exposed conjunctiva, which can serve as a portal of entry.3 the 3 with SARS-CoV RNA in their tears had sampling performed early in their disease course and whether these patients had conjunctivitis was not specified. Although no one studied whether SARS-CoV was present in convalescent patients or whether tears of infected patients were infectious, ophthalmologists should probably don protective equipment when examining certain groups of patients in the pandemic caused by SARS-CoV-2. With the SARS outbreak in 2004, Singaporean ophthalmologists wrote, “Stringent barrier methods using the ‘M3G’  should be the gold standard when dealing with suspected SARS patients.”3 These recommendations make sense in 2020 for ophthalmologists examining high-risk patients. Hand hygiene should occur before and after every patient encounter. Prior to patient examination, ophthalmologists should perform hand hygiene consisting of alcohol-based hand sanitizer or washing hands with soap and water for 30 seconds. Then they should wear gloves, which should be discarded after every patient encounter, followed by hand hygiene. Wearing a mask and eye protection is also very important for ophthalmologists to prevent viral entry via the conjunctiva or respiratory tract.In a 2004 series of 36 patients with suspected SARS-CoV,10 They implemented the following measures: (1) they reduced clinic volume and number of elective procedures via short message service  to patients to postpone nonurgent appointments; (2) they required all ophthalmology personnel to wear face masks; (3) at the clinic entrance, they screened all ophthalmology patients and accompanying persons by infrared thermometers; and (4) they administered questionnaires to afebrile patients inquiring about occupation and travel to affected areas or contact with person(s) traveling to such areas or contact with a suspected or confirmed case within 14 days. They also screened for symptoms of upper respiratory tract infection and acute conjunctivitis. If any answer was affirmative, the patient’s appointment was postponed for at least 14 days. Ophthalmologists performing dacryocystorhinostomy were told to don personal protective equipment when performing nasal endoscopy; general anesthesia cases were re-evaluated given the risk of aerosol generation from endotracheal intubation.10It is better to avoid or decrease exposure than try to contain exposures. At the front end of protection of patients and ophthalmology personnel is instituting screening measures prior to patient arrival. Hong Kong provides one example. Starting January 25, 2020, the emergency response level was activated in all public hospitals, and the 2 hospital ophthalmology departments serving the Kowloon peninsula with a catchment area of 1.1 million people adopted even stricter standards to curb disease transmission in ophthalmology patients.Ophthalmologists face specific occupational risks during this pandemic. At present, conjunctivitis appears to be an infrequent though possibly early manifestation of COVID-19. Contact with an infected patient’s conjunctiva or tears probably is not as likely to transmit disease as contact with aerosolized respiratory secretions. Based on anecdotal reports of SARS-CoV-2 transmission to healthcare professionals without eye protection, the ocular tropism of respiratory viruses, and evidence of SARS-CoV transmission to healthcare workers in the SARS outbreak more than a decade ago, masking and eye protection are important for ophthalmologists. As physicians, ophthalmologists have an essential role in guiding infection control measures to protect other patients, staff, doctors, and the community from unnecessary exposures. Like Dr Li, ophthalmologists may be the first to diagnose a more serious condition than isolated conjunctivitis but more important is the increased risk of transmission given the close proximity of ophthalmologist and patient during ophthalmic examination. And like Dr. Li, without the measures described above, ophthalmologists may face a potentially lethal occupational hazard.