We are reporting a case of a 36 year-old Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) positive hypertensive primigravida with postpartum uterine atony that required emergency subtotal hysterectomy at Saint John Hospital Bucur Maternity Bucharest. The maternity was designated as the Coronavirus Disease 2019 (COVID-19) Maternity for Bucharest and Ilfov County since March 2020. The patient was mildly symptomatic for SARS-CoV-2, infection confirmed with reverse transcription polymerase chain reaction (RT-PCR). The caesarean section was performed and a live male fetus was born, 2630 g and Apgar Score of 9 . Postpartum hysterectomy with adnexal preservation was performed because of uterine atony. The postoperative evolution was favorable. The patient was discharged with her baby 10 days after birth. Given the limited resources, the placenta, the umbilical cord and the uterus were not tested for SARS-CoV-2. The pathology exam revealed that on the maternal side there were specific uterine atony lesions as well as endometrial and miometrial ischaemia. The placenta had nonspecific findings: chronic ischemic lesions with small villi, fibrin deposits in the materno-fetal interface. The peculiarity of the case is that we report the morphological findings of the placenta and uterus resulted from intrapartum uterine atonia in a patient with gestational arterial hypertension, premature birth and COVID-19. Further studies are required to characterize the pattern of such intricate conditions. Coronavirus disease COVID-19) is caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Its outbreak was declared as a pandemic by the World Health Organization (WHO) in March 2020 9 is caus. AllegedThe impact of the virus in pregnant women is of significant interest. In general, pregnant women are usually tested for SARS-CoV-2 infection, on hospital admission for delivery; most of them are often asymptomatic. Typically, the clinical presentations and severity of COVID-19 during pregnancy are similar to the disease spectrum observed in non-pregnant adults; however, some critical cases have been reported ,8.We have presented a case of a 36-year-old woman from Bucharest who was admitted in our clinic for fever and chills that had started three days earlier. The case report was written following The CARE Guidelines: Consensus-based Clinical Case Reporting (CARE) and the patient signed the informed consent for the publication of the data . She wasOn admission, the patient was in a state of general good health, without respiratory symptoms or fever. Uterine contractions occurred at an interval of 2–3 min with minimal cervical changes (the cervix had multiple scarring from the cone biopsy). Ultrasound examination findings revealed oligohydramnios, and the blood test results revealed unremarkable findings. Considering the cervical dystocia and associated morbidities, cesarean section was performed. The newborn male weighed 2630 g and had an Apgar score of 9. The baby tested negative for SARS-CoV-2 at birth and re-tested 8 days after, in line with our testing protocols. After placenta delivery, uterine contraction was absent even after administration of 10 International Units (IU) oxytocin and ergometrine. Heavy bleeding and lack of efficient contractility (uterine atony) required an emergency subtotal hysterectomy with adnexal preservation. The immediate postoperative evolution was satisfactory with antibiotic and anticoagulant treatment, and the patient received 2 units of blood. Pulmonary Rx performed on the third day postoperatively revealed a left laterobasal condensation process and the decision to add hydroxychloroquine  was made by an infectiologist . The patient was discharged with her baby 10 days after birth (with two negative RT-PCR SARS-CoV-2 tests) and made a full recovery.®, Buffalo Grove, IL, USA). Additionally, immunohistochemistry was performed to evaluate the hyperplastic trophoblastic and microvascular processes described in the literature using CD-34 QBEnd/10 and AE1–AE3 polyclonal antibodies on an immunostainer . The corresponding antigens and the counterstain were subsequently stained using Harris hematoxylin. The slides were examined using an Olympus BX 45 microscope with an ICC50W camera , and the slides were scanned using an AperioAT2 turbo station .The uterus, placenta, and umbilical cord were evaluated at the pathology department. On gross examination, the uterus specimen measured 40 cm × 30 cm × 20 cm and weighed 1355 g, with marked areas of congestion and overall low consistency. The placenta had a diameter of 23 cm, and the umbilical cord measured 50 cm. A diffuse deposit of fibrinoid and small adherent blood clots was noted. The specimens were fixed using formaldehyde (4%) according to routine procedures—tissue processing using an automatic Leica Peloris3 station and paraffin inclusion. The resulting slides were hematoxylin and eosin stained on an automatic line , the microscopic findings were variable intumescent islands of smooth muscle cells with pale eosinophilic cytoplasm and pyknotic nuclei-necrotic aspects .The villous stroma appeared fibrotic with a marked vascular congestive appearance, as confirmed by immunohistochemistry .The placental samples showed abundant deposits of fibrin located in the intervillous space, with areas of avascular villi and occasional hemosiderin deposits and calcifications. The villi appeared to be compressed with a small caliber and hyper-vascularity aspects. Notably, we found diffuse eccentrically trophoblastic hyperplasia, with trophoblastic detachments, without macrophagic multinucleated giant cells, neither significant atypia or viral nuclear inclusions .The mother had specific uterine atony lesions as well as endometrial and myometrial ischemia.Placental samples had nonspecific findings such as chronic ischemic lesions with small villi and fibrin deposits at the materno–fetal interface and trophoblastic hypertrophy/hyperplasia and vascular hyperplasia Light-microscopic procedures were shortened.In summary, the analysis of the specimens revealed the following facts:Perinatal transmission of COVID-19 has been reported and discussed widely; however, the precise transmission route  is unclear . In someAsymptomatic infections have also been reported in infants and neonates. In a review of 160 infants with confirmed COVID-19 , 16% infSeveral studies have linked premature births with SARS-CoV-2 infection even in mild cases; however, the possible mechanisms are currently under discussion. Similarly, in our case, premature delivery was performed at 36 weeks of gestation due to SARS-CoV-2 infection, and the involvement of the virus in premature contraction induction can be considered ,19,20.The histopathological findings of the placenta, including chronic ischemic lesions with small villi, fibrin deposits in the materno–fetal interface, trophoblastic hypertrophy and hyperplasia, and vascular hyperplasia could be considered adaptive mechanisms in an environment of chronic ischemia. However, these changes have also been described in other viral infections of the placenta, such as Zika virus infection .Uterine atonia can be theorized to be caused by a combination of chronic maternal hypertension, chronic treatment with nifedipine, SARS-CoV-2 infection, and preterm birth. The significant interstitial edema that dissociated the muscular fibers and the conjunctive matrix can be considered the most important factor leading to uterine contraction insufficiency. Recent studies have proposed the theory of postpartum acute myometritis, which is characterized by specific acute inflammatory changes in the uterine myometrium, such as infiltration of neutrophils and macrophages, increased number of complement C5a receptors in the interstitial area, anaphylactoid reaction, and massive stromal edema; however, the exact cause has not been discovered .This case report highlights that subtle non-specific lesions of the maternal vascular endothelium have important clinical relevance and outcomes. Previous studies have reported these lesions are pseudo-atheromatous, with an intraparietal accumulation of macrophages and fibrinoid necrosis . AlthougGiven the novelty of the virus, evidence regarding the vertical transmission of the virus is scarce and additional studies are needed because of the important implications and severe complications .The present study supports the conclusion of a recent meta-analyses where the authors proved that COVID-19 with at least one co-morbidity increases risk of intensive care and mortality . HoweverCOVID-19 is a systemic disease with various manifestations; the clinical manifestations of this disease in pregnant women, who are routinely tested for SARS-CoV-2 infection on hospital admission for delivery, are of great interest. Although these patients are often asymptomatic, it seems that the virus might have important consequences regarding the way of birth, postpartum recovery, and even fetal outcomes.In conclusion, the manifestations observed in this case were similar to those reported in other cases of maternal SARS-CoV-2 infections. The peculiarity of the case is that we report the morphological findings of the placenta and uterus resulted from intrapartum uterine atonia in a patient with gestational arterial hypertension, premature birth and COVID-19. Further studies are required to characterize the pattern of such intricate conditions.


Cardiovascular death is a common outcome in population-based studies about new healthcare interventions or treatments, such as new prescription medications. Vital statistics registration systems are often the preferred source of information about cause-specific mortality because they capture verified information about the deceased, but they may not always be accessible for linkage with other sources of population-based data. We assessed the validity of an algorithm applied to administrative health records for identifying cardiovascular deaths in population-based data.Administrative health records were from an existing multi-database cohort study about sodium-glucose cotransporter-2 (SGLT2) inhibitors, a new class of antidiabetic medications. Data were from 2013 to 2018 for five Canadian provinces  and the United Kingdom (UK) Clinical Practice Research Datalink (CPRD). The cardiovascular mortality algorithm was based on in-hospital cardiovascular deaths identified from diagnosis codes and select out-of-hospital deaths. Sensitivity, specificity, and positive and negative predictive values  were calculated for the cardiovascular mortality algorithm using vital statistics registrations as the reference standard. Overall and stratified estimates and 95% confidence intervals (CIs) were computed; the latter were produced by site, location of death, sex, and age.The cohort included 20,607 individuals . When compared to vital statistics registrations, the cardiovascular mortality algorithm had overall sensitivity of 64.8% ; site-specific estimates ranged from 54.8 to 87.3%. Overall specificity was 74.9%  and overall PPV was 54.5% , while site-specific PPV ranged from 33.9 to 72.8%. The cardiovascular mortality algorithm had sensitivity of 57.1%  for in-hospital deaths and 72.3%  for out-of-hospital deaths; specificity was 88.8%  for in-hospital deaths and 58.5%  for out-of-hospital deaths.A cardiovascular mortality algorithm applied to administrative health records had moderate validity when compared to vital statistics data. Substantial variation existed across study sites representing different geographic locations and two healthcare systems. These variations may reflect different diagnostic coding practices and healthcare utilization patterns.The online version contains supplementary material available at 10.1186/s12913-021-06762-0. Cardiovascular death is a cause-specific outcome of interest in many studies about the comparative effectiveness of new healthcare interventions. For example, studies about the safety and effectiveness of new prescription medications compared with existing medications frequently use both all-cause and cause-specific death as endpoints , 2. WhenRoutinely-collected, population-based administrative health data, including hospital records and physician visit records, represent an alternative source to identify specific causes of death . AdminisThe aim of our study was to assess the validity of an algorithm applied to administrative health records in multiple jurisdictions for identifying cardiovascular deaths. We used vital statistics registrations as the reference standard to validate the cardiovascular mortality algorithm.Data were from an existing multi-database retrospective cohort study conducted by the Canadian Network for Observational Drug Effect Studies (CNODES) , a pan-CIn each Canadian province, study data included vital statistics registrations, health insurance registrations, physician billing claims, hospitalization records, emergency department (ED) visit records (not available in Manitoba), and prescription drug dispensation records. These data sources can be linked at the individual level using anonymized personal health numbers. Vital statistics registrations capture official records of births, stillbirths, deaths, and marriages. In death records, the underlying cause of death is recorded using the World Health Organization’s International Statistical Classification of Diseases and Related Problems (ICD), 10th revision  . The regStudy data were also obtained from the CPRD, a large UK primary care database containing medical information documented by primary care physicians for approximately 15 million patients enrolled in over 700 general practices . The datThe cohort has been described in detail elsewhere –16. BrieWe constructed our validation cohort from this initial study cohort for those sites where linkage of administrative health records and vital statistics registrations (death registrations from ONS in CPRD) was possible and for those years for which these registration data were available  in-hospital death with a cardiovascular disease diagnosis in the primary/most responsible diagnosis position, or (b) out-of-hospital death (including death in an ED) without documentation of cancer in the 365 days prior to and including the date of death and without documentation of trauma in the 30 days prior to and including the date of death. A significant proportion of all cardiovascular-related deaths are known to occur outside of hospital –24. We sThe list of relevant diagnosis codes to identify in-hospital cardiovascular deaths is provided in Table In vital statistics registrations, which were used to validate the algorithm, cardiovascular deaths were those that had an underlying cause of death with a cardiovascular disease diagnosis. The relevant ICD-10 codes are provided in Table The validation cohort was described using frequencies and percentages. Validity of the cardiovascular mortality algorithm was assessed using sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV). All estimates are reported as percentages.Sensitivity was calculated as the number of correctly-identified cardiovascular deaths in administrative health records divided by the total number of cardiovascular deaths from vital statistics registrations. Specificity was calculated as the number of correctly-identified non-cardiovascular deaths from administrative health records divided by the total number of non-cardiovascular deaths from vital statistics registrations. PPV was calculated as the number of correctly-identified cardiovascular deaths in administrative health records divided by the total number of cardiovascular deaths identified from administrative health records. NPV was calculated as the number of correctly-identified non-cardiovascular deaths in administrative health records divided by the total number of non-cardiovascular deaths identified from administrative health records. The 95% confidence intervals (CIs) were calculated for all estimates; they were based on the binomial distribution.Estimates were produced overall , for the five Canadian provinces, and individually for each of the six sites. Overall and site-specific estimates were also stratified by location of death , sex and age group .As Fig. More than two-thirds of the validation cohort Table  were froSlightly more than half  of the deaths included in the validation study were identified as in-hospital deaths Table . AmongstTable The CPRD produced the highest site-specific estimates of overall sensitivity  and NPV . The Canadian province of Manitoba produced the lowest estimates of sensitivity  and NPV , but the highest estimate of PPV . The province of Quebec had the highest estimate of specificity . The lowest estimate of PPV was for the province of British Columbia .Figure Validity estimates were stratified by sex and age group, respectively  and relevant medications may result in increased accuracy. This finding of increased accuracy has been observed for cardiovascular disease risk predictions from machine-learning algorithms when compared to risk predictions based on conventional statistical models .Cardiovascular diseases are a major cause of death worldwide. A cardiovascular mortality algorithm based on routinely-collected administrative health records is therefore potentially valuable for many population-based studies, including those about comparative effectiveness of new healthcare interventions or treatments, such as new prescription medications. This study found only modest overall validity of the cardiovascular mortality algorithm when compared with vital statistics registrations, but substantial variation in validity estimates across sites. This variation suggests there are opportunities for methodological studies to address the bias associated with using a cardiovascular mortality algorithm derived from administrative health records.Additional file 1. Validity estimates for a cardiovascular mortality algorithm applied to administrative health records stratified by sex and age group. This file contains estimates of sensitivity, specificity, positive predictive value, and negative predictive value stratified by sex and age group .


Intracranial atherosclerotic disease (ICAD) is a dynamic process that leads to ischemic stroke. Symptomatic ICAD patients still suffer a high recurrent rate even under standard treatment. In this case report, to better understand the response of intracranial atherosclerotic plaques to medication, serial MR imaging was added to standard clinical workup in a 47-year-old male patient with acute occipital lobe infarction at baseline, 3-month, 6-month, and 12-month post index stroke to directly visualize the morphology and signal change of plaques. We noticed that one of the plaques showed dramatic worsening at 3-month imaging follow-up despite a decrease in low-density lipoprotein level. Early identification of patients who do not respond well to medication is critical to prevent the recurrence of cardiovascular events in ICAD patients. Intracranial atherosclerotic disease (ICAD) represents a significant risk factor for ischemic stroke worldwide, and patients with symptomatic ICAD experience a high rate of recurrent stroke  V4 segment with luminal irregularities, with concerns for focal dissection, atherosclerosis, or both. This prompted initiation of intravenous heparin. Routine brain MR revealed an acute occipital lobe infarction and a slight increase in pre-contrast T1-weighted signal within the right VA V4 segment. Other laboratory findings included low-density lipoprotein (LDL) of 123 mg/dL and hemoglobin A1c of 5.7%.Due to the uncertainty involved with the lesion on the right V4 segment, further neurovascular imaging including 3D time-of-flight MRA and pre- and post-contrast MR-VWI  and dual antiplatelet therapy (aspirin 325 mg daily and clopidogrel 75 mg daily) was initiated. The patient was hospitalized for 5 days. He was discharged with a minor headache and a National Institutes of Health Stroke Scale score of 0. Two months later, direct LDL level showed a decrease to 95 mg/dL at clinical follow-up. There were no adverse effects from the statin therapy noted. At this point, ezetimibe 10 mg daily was initiated by his cardiologist for further lowering the LDL level. However, statin therapy was stopped thereafter due to miscommunication between the patient and cardiologist.At 3 months after the index stroke, a follow-up MR scan was performed. Despite the moderate regression of LDL and an appreciable drop in plaque-wall CR and/or plaque ER in the two originally identified lesions, a new lesion in the distal right VA with a diffusive and severe lumen narrowing and strong vessel wall enhancement was observed , which led to a drastic reduction in LDL from 67 mg/dL at 6 months to 18 mg/dL at 11 months.MRA and MR-VWI scans were performed at 6- and 12-month post index stroke. Compared with the 3-month scan, patient's 6-month MRA showed a slight regression of lumen at the plaque in the distal right VA, but plaque ER measured on MR-VWI substantially reduced. Twelve months after the index stroke, both degree of stenosis measured on MRA and plaque ER measured on MR-VWI regressed to the baseline level at the plaque in the distal right VA. CR and ER of the other two plaques slightly improved while the degree of stenosis remained unchanged. During this period, the patient's condition has been stable without any new symptoms.The quantitative measurement results of plaque features are shown in Serum lipid biomarkers are widely accepted to monitor ICAD patients' responses to treatment; however, these are systemic rather than lesion-specific markers. MR-VWI can more specifically interrogate each atherosclerotic lesion and provide information beyond the severity of stenosis. Previous studies , and plaque ER (a marker of inflammation)  would further reduce cardiovascular risk  the small size of intracranial vessels, (2) the limited spatial resolution, (3) the lack of additional contrast weighting such as T2-weighting. A high-resolution multi-contrast protocol would make MR-VWI challenging for both patients and clinical workflow.In summary, this case report sheds light on the importance of the promising combination of serum biomarkers and lesion-specific imaging tools in the decision-making of stroke patients.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by Ethics Committee of Cedars-Sinai Medical Center. The patients/participants provided their written informed consent to participate in this study.JX and MP interpreted the data and drafted the manuscript. SS, ZF, and KS contributed substantially to the design of the work and revised it critically for important intellectual content. All authors contributed to the article and approved the submitted version.SS and ZF received salary support from the NIH/NHLBI grant # R01HL147355.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


TM process, heterogeneous integrations of wide-bandgap semiconductors, infrared materials, and electro-optical crystals via wafer bonding technology for thin-film transfer are orderly presented. Furthermore, device design and fabrication progress based on the platforms mentioned above is highlighted in this work. They demonstrate that the transferred films can satisfy high-performance power electronics, molecular sensors, and high-speed modulators for the next generation applications beyond 5G. Moreover, flexible composite structures prepared by the wafer bonding and de-bonding methods towards wearable electronics are reported. Finally, the outlooks and conclusions about the further development of heterogeneous structures that need to be achieved by the wafer bonding technology are discussed.Wafer bonding technology is one of the most effective methods for high-quality thin-film transfer onto different substrates combined with ion implantation processes, laser irradiation, and the removal of the sacrificial layers. In this review, we systematically summarize and introduce applications of the thin films obtained by wafer bonding technology in the fields of electronics, optical devices, on-chip integrated mid-infrared sensors, and wearable sensors. The fabrication of silicon-on-insulator (SOI) wafers based on the Smart Cut TM technology , has been proposed and used for the fabrication of high-quality single-crystal thin film onto different substrates. Correspondingly, the application of wafer bonding technology has been dramatically expanded. Among them, Si-on-insulator (SOI) wafers that have been widely used in various electronic products and photonic devices are one of the most successful commercial cases. The top Si thin film can serve as the functional layer for both electrical and optical designs. With the rapid rise of new energy vehicles, wafer bonding technology of wide-bandgap semiconductors, such as gallium nitride (GaN), silicon carbide (SiC), and gallium oxide (Ga2O3), for power electronics is gradually being developed and improved [Wafer bonding is a popular technology that can integrate two or more kinds of materials into one heterogeneous system. It has been successfully applied to system-in-package (SiP) and fabrication of heterogeneous structures ,4,5,6,7.improved ,12,13. D3) thin film has a significantly high electromechanical coupling. However, it is currently not possible to achieve the preparation of LiNbO3 film on the substrate/insulator through conventional deposition processes. Exploiting the transfer technique to move a slice of the bulk LiNbO3 and bond to a carrier substrate is the most common and effective method [3 electro-optic modulator has shown outstanding advantages in terms of the speed of information transmission and bandwidth modulation [3-on-insulator (LNOI) platform, wafer bonding provides effective technical support for the on-chip heterogeneous integration of LiNbO3 thin film. This idea has been confirmed to be feasible by designing and preparing high-speed modulators on LNOI wafers, which are fabricated using the wafer direct bonding technology to peel off the single-crystal thin film from the bulk LiNbO3 crystals [Currently, we are on the cusp of communication technology innovation. The development of the fifth-generation (5G) communication technology and alternative communication technologies beyond 5G will affect the technology landscape of countries worldwide. The communication technologies aim to be a revolutionary leap forward in data rates, latency, massive connectivity, network reliability, and energy efficiency ,15,16,17e method ,19. Movidulation . As for crystals ,22. This2). Then, thin films can be transferred onto flexible substrates using the combination of sacrificial removal processes by hydrofluoric acid solution and hot-press bonding processes. Due to the bonding and de-bonding processes compatible with the complementary metal-oxide-semiconductor (CMOS) technology can provide large-scale and stable substrates for subsequent device fabrication. Meanwhile, in the era of big data and artificial intelligence, next-generation wearable sensors will enable human beings to interact with other objects in more and more applications. Driven by such trends, the Internet of Things (IoTs) and artificial intelligence of things (AIoTs) are indispensable considerations for future sensor design and development [In addition to the above mentioned on-chip integrated applications, thin-film transfer relying on the combination of bonding and de-bonding methods can also be used to fabricate wearable electronics. With the continuous improvement of life quality for human beings, wearable sensors and human-machine interfaces have been considered essential for future multi-functional sensors related to human health ,26,27,28elopment .TM process, including wafer bonding and ion implantation processes. Additionally, applications of different platforms in the on-chip integration are presented. Secondly, the different wide-bandgap semiconductor thin-film transfer progress based on the wafer bonding methods for high-power electronics is reported. Thirdly, achievements of LiNbO3 thin-film transfer for high-speed electro-optical modulators towards the next generation of communication technology are summarized. Fourthly, film transfer of infrared materials and their applications in molecular sensors are systematically introduced. Finally, we emphasize the methods on the functional thin-film transfer onto flexible substrates for the next generation of wearable sensors, which are related to human health monitoring.In this review, we summarized the high-quality thin films fabricated using wafer bonding technologies and transferred methods in different fields. Divided by materials, we reviewed electronics devices, optoelectronics devices, on-chip integrated mid-infrared sensors, and wearable sensors for communication and sensing applications. The schematic diagram of the article structure is shown in TM technology) has been developed [Semiconductors are fundamental for all kinds of photonic and electronic components. They are the basic platform for device design and fabrication. Si- and Ge-based materials are the most popular among numerous semiconductors due to the advances in mature fabrication, low cost, and mass production ,31. Thereveloped ,33. ThisTM technology. The standard Smart Cut process for the SOI fabrication is shown in +) implantation creates a destroyed layer  on the subsurface. There are several advantages to implant the light element  into the donor wafers. The transferred layer can be engineered with no defects. By controlling the implantation energy of H+, the thickness of the transferred layer can be determined with a high degree of precision. After finishing the ion implantation, the oxidized wafers are bonded together via an activated bonding method. Then, an annealing process is performed on the bonded pairs. On the one hand, the donor wafer will be completely cracked from the bubble layer, making the thin film peel off and remain on the supporting wafer. On the other hand, high-temperature treatment will be beneficial for strengthening the bonding interface between the thin film and supporting substrate. Finally, chemical-mechanical polishing (CMP) is used for flattening and smoothing surfaces to satisfy practical production requirements. For the donor wafers, substrates can be refreshed and reused after each layer transfer operation. The cross-section image of the fabricated SOI wafer is shown in Today, most industry-leading Si-on-insulator (SOI) wafers destined for chip manufacturing are made by Smart Cut2 wafer and SOI wafer are the upper and lower layers of the device, respectively. Microstructures patterned on the two parts are fabricated separately and assembled via the flip-chip bonding method. The optical waveguide couplers are prepared on the bottom SOI wafer, including suspended subwavelength grating (SWG) waveguides, grating couplers, and a tunable waveguide coupler with a MEMS cantilever. To make the MEMS cantilever move up and down, a single-side cladding for the suspended SWG waveguide is used to replace double-side cladding in the cantilever region. Leveraging the advantage of this change, the movement of the MEMS cantilever and optical signal transmission along the waveguide can be simultaneously realized with less crosstalk. The Au pad is fabricated on the top SiO2 wafer and covers the cantilever region. Al solders are used for the intermediate bonding of the top SiO2 wafer and bottom SOI wafer and the spacers and electrical interconnections. The MEMS cantilever can be actuated by applying a voltage between the top and bottom electrodes. The light propagates in the waveguide and couples from the input-port to drop-port in the lack of voltage bias. Correspondingly, the power-splitting ratio can be continuously tuned in the range of 0% and 100%. Based on the advances of the SOI platform and design, an optical attenuator with 25 dB depth using DC voltage actuation has been realized. With the actuation of AC voltage, the 1 × 2 optical switches with a response time of 8.9 μs and −3 dB bandwidth up to 127 kHz has been demonstrated.Recently, a typical multifunctional photonic switch on the SOI platform operating in the mid-infrared (MIR) wavelength range (from 3.85 μm to 4.05 μm) has been reported . They usDue to the excellent infrared properties of Si, another important application for the SOI platform is used for the photodetectors combining with two-dimension materials  . A desig1−xSnx) alloy is a famous Group IV semiconductor for potential applications in both electronics and photonics [1−xSnx can be tuned. The combination of Group IV-based optical components with high-speed CMOS circuits can be desirable in future integrated chips. Therefore, the high-quality Ge1−xSnx layer on the insulator is an essential platform for monolithic integration. Currently, Ge1−xSnx thin film with a surface roughness of 0.528 nm can be grown on the (001)-oriented Si substrate using a molecular beam epitaxy (MBE) system. To fabricate the Ge1−xSnx on insulator structure, a 500 nm-thickness SiO2 layer is deposited on the Ge1−xSnx using the PECVD. To make the SiO2 denser, a 350 °C thermal treatment for 7 h in an N2 environment is conducted. Subsequently, the CMP process is performed on the SiO2 surface to reduce the surface roughness. The root-mean-square (RMS) value smaller than 0.2 nm has been achieved, which can satisfy the requirement for wafer direct bonding [2/Ge1−xSnx/Si and Si supporting wafer surfaces are activated by the O2 plasma for 15 s and contacted at room temperature. A post-annealing at 300 °C in an N2 environment is carried out to further enhance the bonding strength. Finally, the top Si is removed by soaking the bonded pair into tetramethylammonium hydroxide (TMAH) solution with an 80 °C heating treatment, while the bottom Si supporting wafer is spin-coated by a protective layer to avoid being etched. The cross-sectional TEM image of the obtained GeSnOI is shown in 1−xSnx layer is 95 nm which is the same as the one grown by MBE, revealing the high etching selectivity of TMAH for the Si and Ge1−xSnx.Germanium-tin , as well as highest m,int/SsatG are realized. The result of the comparison for S in GeSn p-FET is shown in To characterize the chemical bonds and strain of the fabricated GeSnOI wafers, Raman spectra are used for the component analysis, as shown in TM technology, it is possible to integrate thin film of III-V compounds onto the Si-CMOS platform. In addition to compound semiconductors, wide-bandgap materials, such as Ga2O3, are also popular in optoelectronic devices.As mentioned above, Si-based CMOS is one of the most fundamental components in semiconductor products. Driven by Moore’s Law in the past year, device miniaturization and multifunction are the goals of chip development and pursuit. As the feature size continues to scale down, many advantages, such as improved operation speed, reduced power consumption, and production cost, gradually emerges ,76,77. UTM technology. However, the generated gas originated from the InP/Si bonding will cause unavoidable bubbles in the interface, reducing the usage of integrated heterogeneous wafers. A modified method using surface trenches to eliminate interfacial voids has been proposed [TM technology for InP thin-film transfer is shown in 2 layer using photolithography followed by the plasma etching process. By optimizing the parameter of trench space, the bubbles can be efficiently inhibited. To highlight the effect of trenches, half of the SiO2-on-Si wafer with channels and a half without channels are used for InP heterogeneous bonding. The bonding results after annealing are shown in 2/Si bonding interface across the trench region is tight. TEM observation also confirms that the atomic bonding between InP and SiO2 has been achieved. Relying on this modified method, InP thin film with 2-in can be successfully transferred onto the SiO2-on-Si wafer. The whole area of the InP/SiO2/Si heterogeneous substrates is uniform, flat, and high-quality, can be used for practical device fabrication. We should note that the lateral outgassing surface trenches (LOTs) mechanism to facilitate the generated gas diffusion out of the bonding interface is a physical effect, only relying on the design of LOTs. This modified Smart CutTM technology might be a promising method to remove the bubbles at the bonding interface for all III-V compound semiconductors when integrated with the SiO2, SiO2-on Si, or Si substrates.The InP thin-film transfer via wafer bonding is similar to the Smart Cutproposed . The schTM process [2/Si bonding interface can keep stable during the post-annealing to peel off the GaN thin film from the bulk GaN wafer. The optical image of the transferred GaN-on-insulator (GaNOI) wafer is shown in Gallium nitride (GaN) is an ideal material to apply in wireless communications and optical communications because it can operate in high power and frequency ,92,93. T process . The sch2O3 with ultra-wide bandgap has been considered a promising material for next-generation power electronics [2O3 as the intermediate layer to perform the Smart CutTM process [2O3/Si heterogeneous structure can be used for the fabrication of MOSFETs. The schematic structure of the proposed Ga2O3 MOSFET on (001)-oriented Si substrate is shown in 2O3 thin film and SEM image of the MOSFET microstructure is presented in brV above 600 V is achieved at 500 K, which is weakly dependent on the temperatures. It shows a significant improvement in thermal stability compared with the Ga2O3-on-SiC devices. Therefore, it proves that thin-film transfer via wafer bonding is a promising method to overcome the thermal limitation in Ga2O3 power electronic applications.In addition to GaN semiconductor, Gactronics ,101,102. process . The tra2O3 are mainly high-power or high-frequency electronic devices because of their excellent material properties [Although the heterogeneous integration of wide-bandgap semiconductors and Si-CMOS can bring many benefits, applications of GaN and Gaoperties ,104. Howoperties ,108,109.operties ,111. Theoperties ,118,119.TM technology, the laser lift-off is used for the GaN thin-film transfer. The schematic diagram of the experimental process is shown in To achieve a reliable bonding between GaN and SiC, the SAB method has been used for direct wafer bonding . Differe2O3 [2O3 thin-film transferred procedure is the same as the Smart CutTM process, as shown in 2O3 layers as the intermediate for the modified SAB method. Optical images of the Ga2O3/SiC bonded wafer pair and transferred Ga2O3 thin film are shown in 2O3 and SiC has been achieved bridging by Al2O3, as presented in 2O3/SiC wafer, as presented in 2O3/SiC heterogeneous wafer is reduced to 130 arcsec, demonstrating the high crystallinity of the Ga2O3 layer.Not only limited to the GaN, the SAB method for the thin-film transfer is also suitable for the Ga2O3 ,121,122.3 (KNN) is more friendly to the human body and environment because it is lead-free, lead zirconate titanite (PZT) has more advantages in the performance and fabrication of implanted devices due to the higher piezoelectric effect [Implantable electronics have important research significance for human health detection, disease diagnosis, and treatments. Several intelligent automatic devices have been applied in clinical practice to improve patients’ health. Although many self-powered devices, such as triboelectric- or electrochemical-based devices, can detect the human body’s dynamics, they cannot be applied in-vivo because of the limited biocompatibility of materials ,132,133.c effect ,143,144.2 layer [2-on-Si wafers by the CMOS-compatible process. The device structures have also been transferred at the same time. Secondly, the photoresist is spin-coated on the wafer surfaces to prevent the device structure from being corroded by subsequent HF solutions. After the immersion treatment, the deposited PZT thin film with photoresist protection will be separated from the SiO2-on-Si wafer. Finally, the functional thin film will be stamped by the PDMS onto the PI substrate for the implantable electronics. Based on this process and composite structures, implantable devices that enable high-efficiency mechanical-to-electrical energy conversion have been proposed. The device parameters and structures are shown in The PZT thin film transfer onto different flexible substrates is based on the debonding method of hydrofluoric (HF) acid to remove the SiO2 layer . The finThe same process can also be used for the piezoelectric composite thin film transfer onto the PDMS substrate . RecentlMoreover, the biocompatibility of the iPEG has also been tested, as shown in 3 is a popular material due to its excellent piezoelectric property, electro-optic and nonlinear optical effects. However, traditional bulk LiNbO3-based optical devices suffer from large volume, expensive, low bandwidth, and high operating voltages. To overcome this problem, thin-film LiNbO3 is a good choice. Currently, there are already several kinds of commercial LiNbO3 thin-film wafers, such as LiNbO3-on-Si, LiNbO3-on-sapphire, LiNbO3-on-glass, etc. Fabrication of the above LiNbO3-based heterogeneous wafers is based on the Smart CutTM technology [+ implantation is performed to create a damage layer in the LiNbO3 sub-surface. After surface activation, the activated surfaces of LiNbO3 and handle substrate are contacted to form the pre-bonded pair. With an annealing treatment, the donor LiNbO3 wafer is cracked from the damage layer. At the same time, the bonding interface between the LiNbO3 thin film and handle wafer will be strengthened because of the higher temperatures. After the CMP process, heterogeneous wafers can be used for device fabrication. However, LiNbO3 is a kind of chemically inert material. The large propagation loss caused by the rough sidewalls is due to the imperfect fabrication. A rib configuration for the LiNbO3-based optical devices has been proposed to better confine the waveguide light, as shown in 3 can satisfy the requirements of high-performance EO devices. Recently, a 50:50 Y-junction Mach-Zehnder interferometer and an EO comb generator have been proposed, as shown in 3-based EO comb generator, the frequency range covering the entire telecommunication L-band has been achieved. Both demos demonstrate that the LiNbO3-based EO devices have huge potential in applying 5G and even the next generation of communication technology.LiNbOchnology . The pro3 thin film has been proposed [3 are shown in 3-based EO transducer shows system simplicity and low-noise operation, showing opportunities for the on-chip filter integration and further improvement of the transduction efficiency.Transducers are promising platforms that may enable large-scale quantum networks because of the direct conversion, low-noise operation, and large bandwidth. A superconducting cavity EO transducer based on the LiNbOproposed . The schn = 3.48) and SiO2 (n = 1.40), the existence of SiO2 limits the operating wavelength range of Si photonics because of the light absorption. To make Si-based devices have better performance when the working wavelength is larger than 4 μm, transferring Si thin film to infrared substrates with lower refractive indexes, such as sapphire, MgF2, CaF2, and BaF2, is a good choice.There are many fingerprints for gases, liquids, and biomolecules in the MIR wavelength ,158,159.2 substrate is shown in 2 layer. Due to the existence of photoresist, the underlying cured and baked photoresist become pedestals to support the top Si layer on the supporting substrate after completely releasing the BOX. Subsequently, a polydimethylsiloxane (PDMS) film with a thickness of several micrometers is used to stick on the surface of the released top Si thin film. We should note that the contact between PDMS and Si thin film mainly depends on the adhesion force, which can be regarded as temporary bonding, but the bonding strength is stronger than the fracture strength of the photoresist. Therefore, the Si membrane can be peeled off from the SOI platform. After that, the Si/PDMS composite is pressed on the receiving CaF2 substrate, and PDMS is slowly removed. Consequently, the Si thin film is bonded onto the CaF2 substrate with the assistance of surface forces. Although the bonding of Si and CaF2 is under the effects of van der Waals and other temporary bonds, experiments have verified that the substrate can withstand the erosion of chemical solutions.The primary fabrication process for developed Si thin-film transfers onto the CaF2 substrate has been fabricated and published [2 substrate. It can be seen that the surface is flat and smooth. The microstructures of the Si microring resonators coupled with bus waveguides are shown in The above-mentioned method is capable of transferring Si thin film with an area of several square centimeters. Based on this process, the first Si photonic device integrated on the CaFublished . Figure 2 molecules have been introduced for measurement. A dip recovering with a full width at half maximum of 170 nm at 4.26 μm has been observed. More high-performance passive or active miniaturized MIR devices will be developed based on the technology of Si thin-film transfer onto the CaF2 substrate.The heterogeneous integration of waveguide photodetectors towards chip-scale zero-bias long-wave infrared (LWIR) spectroscopic sensing has also been demonstrated . The schTM technology. In such situations, bonding is more critical to achieving reliable fabrication for electronic and photonic devices. However, for the thin-film transfer printing process at the wafer level, debonding is more critical to obtain flexible substrates with large size and fewer surface damages. In this part, we introduce two kinds of technologies  to fabricate high-quality thin-film substrates.Flexible and wearable electronics are an inevitable trend in the development of real-time and dynamic monitoring for human health since the thin-film electronics can offer better performance than traditional bulk systems, being lightweight, low-cost, and visible ,179,180.x layer sandwiched by the Au foil and Si template is generated due to the photoelectrochemically oxidizing. Then, a polymer adhesive is attached to the Au surface for the subsequent thin-film separation. By immersing the composite layer in the hydrofluoric acid solution, the SiOx layer is removed to achieve the peeling. The interfacial microstructures in each step are shown in 2 have been confirmed by TEM. A 28 nm-thickness Au foil with a sheet resistance of 7 ohms per square shows only a 4% increase after 4000 bending cycles. Besides, the grown ZnO nanowires also show flexibility with the nanowires intact up to 500 bending cycles. X-ray diffraction and pole figures have been used to study the in-plane and out-of-plane orientation, as shown in Nano-thickness metal films possess excellent properties in optical transmittance, flexibility, and sheet resistance. Most of them are generated on the substrates by evaporation, sputtering, or other deposition methods, making the thin films polycrystalline. However, the grain boundaries often make electronics suffer from electron-hole recombination, which will degrade their performance. To overcome this problem, introducing a single-crystal Au film on the Si surface as a substrate for epitaxial growth of other single-crystal metal thin films has been developed . The sch2/Si wafer to serve as the separation and glue layer, respectively. With water molecules’ assistance, the glue layer dissolves, and the device layer peels off from the substrate. Additionally, the SiO2/Si wafer can be repeatedly used. Optical images of the n-doped Si thin-film capacitor arrays on a thermally releasable tape, and device performance test are shown in The schematic illustrations of critical steps for the thin film transfer based on the water-induced layer separation are shown in In the past few decades, wafer bonding technology has made remarkable achievements in SiP and the fabrication of heterogeneous structures for electronics. Although it is only one step in developing various platforms, wafer bonding is one of the most essential and indispensable steps in obtaining reliable interfaces among device layers and substrates. Thanks to the extraordinary performance by thin-film, the micro/nano devices show great enhancement in many applications of electronics devices, optoelectronics devices, infrared nanophotonic sensors, and wearable sensors compared with their counterpart made by bulk materials. Although the cost for the wafer increases at the current stage, the gain of device performance is requesting the industry to lower down the cost by developing mature processes for mass production. With the rapid development of 5G technology and IoTs, the fabrication of new heterostructures suitable for various functional devices ushered in both challenges and opportunities. Compared with the developments of bonding methods for semiconductors, more work needs to contribute to infrared material bonding. With the emerging rise of metasurfaces in molecular sensing, more heterogeneous composites for infrared materials need to be developed to satisfy the theoretical research and experimental requirements. In the future, wearable and multifunctional system-level integration combined with extensive data analysis and artificial intelligence is an inevitable trend for sensing applications. Wafer bonding is one of the feasible and effective methods for such a large and complex heterogeneous integration.


Within only one year into the pandemic, several vaccines were designed and reached approval for the immunization of the world population. The remarkable protective effects of the manufactured vaccines are demonstrated in countries with high vaccination rates, such as Israel and UK. However, limited production capacities, poor distribution infrastructures and political hesitations still hamper the availability of vaccines in many countries. In addition, due to the emergency of SARS-CoV-2 variants with immune escape properties towards the vaccines the global numbers of new infections as well as patients developing severe COVID-19, remains high. New studies reported that about 8% of infected individuals develop long term symptoms with strong personal restrictions on private as well as professional level, which contributes to the long socioeconomic problems caused by this pandemic. Until today, emergency use-approved treatment options for COVID-19 are limited to the antiviral Remdesivir, a nucleoside analogue targeting the viral polymerase, the glucocorticosteroide Dexamethasone as well as neutralizing antibodies. The therapeutic benefits of these treatments are under ongoing debate and clinical studies assessing the efficiency of these treatments are still underway. To identify new therapeutic treatments for COVID-19, now and by the post-pandemic era, diverse experimental approaches are under scientific evaluation in companies and scientific research teams all over the world. To accelerate clinical translation of promising candidates, repurposing approaches of known approved drugs are specifically fostered but also novel technologies are being developed and are under investigation. This review summarizes the recent developments from the lab bench as well as the clinical status of emerging therapeutic candidates and discusses possible therapeutic entry points for the treatment strategies with regard to the biology of SARS-CoV-2 and the clinical course of COVID-19.Since November 2019 the SARS-CoV-2 pandemic has caused nearly 200 million infection and more than 4 million deaths globally (Updated information from the World Health Organization, as on 2 The namovirales . Alongsiovirales , 5. The ovirales . The encSARS-CoV-2 is mainly transmitted by aerosols and infects the cells in the upper respiratory tract . HoweverIn general, the immune response to SARS-CoV-2 infection can be broadly described in three phases . The earEven though all age groups can be infected by SARS-CoV-2, particularly elderly patients have an increased risk of mortality. Comorbidities such as obesity, hypertension, chronic obstructive pulmonary diseases (COPD), diabetes, cardiovascular and cerebrovascular diseases have been reported as risk factors for severe COVID-19 , 17. TheClearly, these dynamics in viral replication and immune activation as well as the patient’s individual constitution require specified therapeutic strategies that are aligned to the disease phase and time of treatment onset. Here, we summarize the current state of development and experiences from first in-human applications of the many antiviral and immunomodulatory drugs that are under investigation in single or combinatorial approaches for the treatment and prevention of COVID-19.According to their essential enzymatic functions for the replication of SARS-CoV-2, the S protein, the viral polymerase as well as the viral proteases are the most prominent targets for diverse virus-targeting antiviral strategies.Umifenovir (Arbidol) has been previously applied against some enveloped and non-enveloped viruses especially Influenza A and B, and recently against hepatitis C virus (HCV) . Althougin vitro, ex vivo and in vivo systems -approved anti-leprosy drug and has been shown to reduce SARS-CoV-2 infection by inhibiting spike-dependent cell fusion and the activity of the viral helicase in many  systems . InteresLike for other virus infections, the therapeutic value of convalescent sera that contain neutralizing antibodies to SARS-CoV-2 was evaluated. Although early clinical studies demonstrated improvement of symptoms and viral clearance within an average of 5-6 days in patients receiving convalescent plasma treatment –32, resuAnother antibody cocktail named REGN-COV2 contains the two non-competing neutralizing antibodies Casirivimab and Imdevimab, both targeting the S protein RBD . Resultsin vitro  is an orally available nucleoside analogue with similar mechanism to Favipiravir and inhibits effectively SARS-CoV-2 in  in vivo , 68. It  in vivo . MolnupiLPV/r is a broad-spectrum protease inhibitor used in the treatment of AIDS. Due to its low bioavailability when taken orally, it is used in combination with the pharmacokinetic enhancer ritonavir. LPV/r is supposed to target the SARS-CoV-2 C-like proteinase (3CLpro) substrate binding site, which is highly conserved among all coronaviruses . DespiteAll viruses, including SARS-CoV-2, exploit the host cell resources to replicate and ensure transmission to other hosts. Virus-host interactions occur at all stages of the viral life cycle and provide numerous factors that are crucial for virus survival and therefore may represent targets for antiviral approaches. A major advantage of targeting the host rather than the virus is the low risk of resistance development and the high potential of broad use against other pathogens that rely on related cellular proteins or processes.in vitro, and also in mice using the common-cold corona virus OC43  such as the antidepressant Fluoxetine to inhibit SARS-CoV-2 cell entry has been investigated by several groups. This revealed, that Fluoxetine treatment efficiently reduced viral entry and replication without cytotoxic effects in vitro , 97. A fin vitro . In addiin vitro . On thisin vitro . A retroin vitro . Furtherin vitro. Moreover, SARS-CoV-2 titers in lungs of mice expressing human ACE2 were significantly reduced upon prophylactic treatment with Plitidepsin, facilitated by eEF1A inhibition , called Plitidepsin (Brandname: Aplidin) was discovered and characterized to have potent antiviral effects against SARS-CoV-2 –104. Inthibition . Furtherhibition . Plitidehibition . Intereshibition . This prin vitro and in vivo and thereby could help to counteract or rebalance the SARS-CoV-2 induced hyperactivation of the immune response  or IFN-β1a (IRCT20151227025726N12) with LPV/r on mild-moderate COVID-19 hospitalized patients. The multicentre trial reported that the early triple cotherapy involving systemic administration of IFN-β1b with LPV/r was superior and safe compared to LPV/r alone, in attenuating the disease symptoms, and in reducing the clinical duration of viral shedding and hospital admission . SimilarOnly two associated clinical trials were performed on severe COVID-19 patients to assess the clinical benefits of IFN-β as therapeutic additive to standard medications (HCQ plus LPV/r or atazanavir/ritonavir) and reported favorable clinical outcomes. The trial administering IFN-β1b (IRCT20100228003449N27) reported that the test group had a shorter time to clinical improvement and discharge day and a lower 28-days mortality rate than the control group not receiving IFN-β1b . AlthougThe timing of IFN-administration is likely a strong influential factor on the disease trajectory of COVID-19. Therefore, a retrospective multicenter cohort study carried-out in COVID-19 patients investigated the association between the timing of IFN-α2b administration with its clinical outcomes through regression analysis . ImportaAlthough more Type I IFNs exist in humans, currently only IFN-β (IFN-β1a and IFN-β1b) and IFN-α2 (IFN-α2b) as well as some engineered forms of IFNs are of clinical relevance. However, recent studies investigated the activity against SARS-CoV-2 of all twelve existing IFN-α subtypes, and demonstrated highly efficient and subtype-specific antiviral properties , 138, whin vitro and in vivo  on mild COVID-19 patients tested the therapeutic efficacy of aerosol inhalation of IFN-κ with Topical Trefoil Factor Family-2 (TFF2), which is a peptide isoform demonstrating proven therapeutic benefits against gastrointestinal disorders associated with mucosal damage . This trWhile the concept of cotreatment classically revolves around the use of two different drugs, or currently with a drug combined with an IFN-type, to accomplish improved clinical benefits at lower doses of the individual agents, encouraging results came out of clinical trials evaluating combinations of two different IFN types. For instance, based on the synergic inhibition of SARS-CoV-2 replication by type-I and type-II IFN combinations –149, a cWhile several trials evaluated the therapeutic benefits of IFN administration, preliminary trials are also being deployed on a prophylactic setting to prevent infections. Inspired by the preclinical studies on the high maintenance of IFN concentration as nasal droplets , 151, a Because the development of COVID-19 is strongly driven by a dysregulated, often overshooting immune response, different immune modulatory treatments are considered for early and late phases of COVID-19 and several promising approaches for immuno-therapeutic treatments are under clinical investigations.Glucocorticoids such as Dexamethasone constitute a frequently used class of anti-inflammatory drugs and have been used to treat chronic rhinosinusitis, chronic respiratory diseases or autoimmune disorders like rheumatoid arthritis , 154. ApThe proinflammatory cytokine IL-6 is one of the hallmark cytokines upregulated during severe COVID-19 and represents a predictive marker for the need of mechanical ventilation in accordance with CRP levels . Based oth of June 2021, the FDA gave an EUA for Tocilizumab (Actemra) for the treatment of hospitalized pediatric or adult COVID-19 patients, when they are in need for supplemental oxygen, mechanical ventilation or ECMO. Later, at the 6th of July 2021, the WHO officially recommended the usage of IL-6 receptor blockers based on a network meta-analysis with data from clinical trial investigators in 28 countries (WHO).Tocilizumab and Sarilumab are both monoclonal antibodies, which inhibit the membrane-bound and the soluble form of the IL-6 receptor . TocilizSiltuximab is a human-mouse chimeric monoclonal antibody against IL-6, which is approved for treatment in HIV-negative patients with multicentric Castleman’s disease . Prelimiin vitro (in vitro (The mitogen-activated protein kinase (MAPK) p38 is associated with dysbalanced inflammatory processes in different pathologies and well-studied in animal models of acute lung and myocardial injury , 180. Mein vitro , 183 andin vitro . Based oin vitro . In AuguThe Janus Kinase (JAK) family members play an essential role for the intracellular cytokine receptor signaling . They clSimilar to Baricitinib, the JAK inhibitor Tofacitinib is also approved as an anti-rheumatic drug . When apIn June 2021, a clinical trial using JAK inhibitor Ruxolitinib for treatment of COVID-19 patients was completed (RUXCOVID NCT04362137). Ruxolitinib is already an approved medication for myelofibrosis since 2012 . UnfortuBased on the rationale that the reduction of the overly activated proinflammatory response during COVID-19 improves clinical outcomes, many studies investigate approaches to target proinflammatory processes and other cytokines. Similar to the mechanism of IL-6 antagonists, antagonists of the IL-1 family and TNF blockers were investigated. The IL-1β antagonist Anakinra could successfully ameliorate the cytokine storm in infected patients with severe sepsis . A complIn addition to the treatment of acute COVID-19, the need for efficient therapeutic options for the long-term consequences of the infection rapidly increases as many infected individuals suffer from Long COVID . ReportsWhilst this condition was first described as post COVID-19 syndrome or chronic COVID-19, the patient-derived name Long COVID or Long-haul COVID has recently been favored within the literature to draw attention to its severity , 208. ThThe knowledge of Long COVID is still rather small and it is not even clear if all reports of long-lasting effects on patient´s health describe the same entity . There avia an online questionnaire with 900 participants. In 56.7% of all participants, symptoms overall improved after vaccination, while 18.7% experienced a deterioration. Only in 2.9% of participants all symptoms worsened. Interestingly, the Moderna mRNA vaccine appeared to be especially beneficial in relieving symptoms .To gain more insight into Long COVID the post-hospitalization study (PHOSP-COVID) has been set up in the UK and will recruit 10.000 patients that have been hospitalized with COVID-19, in order to better understand the short-, medium- and long-term consequences of infection More than one and a half years into the SARS-CoV-2 pandemic, the treatment options for severe COVID-19 are still very limited. However, as this review highlights, the number of new drug candidates that are in clinical development or have already received EUA and are on the way to full approval is constantly growing. This include the immunomodulatory JAK inhibitor Baricitinib and the IL-6 receptor blocker tocilizumab, which both targets prevent signaling pathways that are involved in the expression levels of proinflammatory cytokines. The success of such approaches in the treatment of COVID-19 emphasizes that immunomodulatory and not primarily antiviral treatments are the key to COVID-19 therapies as the late-stage clinical manifestation of this disease is dominated by imbalanced inflammatory responses rather than by virus replication itself Figure 1However, antiviral approaches aiming to reduce viral replication are similarly required to prevent the development of COVID-19 which is most successful in the earlier stages of the disease and can prevent further systemic dissemination of the virus and development of systemic inflammation. Under these considerations, combination of antiviral and immunomodulatory drugs or drugs that comprise both actions offers the broadest treatment window and appears most promising to achieve positive clinical outcomes in COVID-19 patients.Clearly, the majority of the new therapeutic options are constituted of repurposed drugs or derived from classical therapeutic strategies such as treatment with convalescent sera or monoclonal antibodies, that are already in clinical use for other diseases and for which the route of administration, safety and side effects in humans is already well characterized. Nevertheless, as this review summarizes, there are also novel strategies, such as the ACE2-Fc fusion protein. Next to clinical therapeutics, home-treatment options are also being assessed for several drugs with the aim to reduce the time of viral shedding and the burden of milder disease. Another advantage of an increased arsenal of treatment options against SARS-CoV-2 and COVID-19 can be seen in potential to prevent the development of long-term complications like Long COVID.LB is responsible for the concept and structure of the review. SK, DC, JJ, AF, BC, and JL collected the literature articles and drafted the individual sections. SK edited the initial draft and designed the final figure. LB and SL reviewed and revised the final draft. All authors contributed to the article and approved the submitted version.German Research Foundation (DFG) through the Clinical Research Unit CRU342 (for Project-P6 to LB and SL); the Federal Ministry of Education and Research (BMBF) for the project CoIMMUNE (Grant No. 01KI20218 to LB), and through the Network University Medicine (NUM) for the COVID-19 project Organo-Strat (Grant No. 01KX2021 to LB and SL); and by the Faculty of Medicine, University of Muenster, through the Interdisciplinary Centre for Clinical Research (IZKF) (Grant No. Bru2/015/19 to LB) and the Innovative Medizinische Forschung (IMF) (Grant No. BR111905 to LB). DC is financially supported through a Study Abroad Program by the Ministry of National Education (Law: 1416), Republic of Turkey. This work was financially supported by the SL is a founder, shareholder and board member of Atriva Therapeutics GmbH, Tübingen, Germany, a pharmaceutical company developing novel HTA against respiratory viral diseases including COVID-19.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Evidence suggests that pregnant women are at a higher risk of complications compared to the general population when infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the reasons that lead them to need intensive care are not clear. This is a prospective multicenter study of SARS-CoV-2 positive pregnant women, registered by the Spanish Obstetric Emergency Group, with the objective to define the characteristics of the mothers who were admitted to the Intensive Care Unit (ICU) and to investigate the causes and risk factors for ICU admission. A total of 1347 infected pregnant women were registered and analyzed, of whom, 35 (2.6%) were admitted to the ICU. No differences in maternal characteristics or comorbidities were observed between ICU and non-ICU patients, except for in vitro fertilization and multiple pregnancies. The main causes of admission to the ICU were non-obstetric causes  and a combination of coronavirus disease 2019 (COVID-19) symptoms and obstetrical complications (31.4%). The multivariable logistic analysis confirmed a higher risk of ICU admission when pre-eclampsia or hemorrhagic events coexist with pneumonia. The incidence of thromboembolic events and disseminated intravascular coagulation were also significantly higher among patients admitted to the ICU. Therefore, surveillance and rapid intervention should be intensified in SARS-CoV-2 infected pregnant women with the mentioned risk factors and complications. Emphasis should always be placed on anticoagulant therapy in these patients due to the increased thromboembolic risk, C-section surgery and immobilization in the ICU. Severe acute respiratory syndrome due to coronavirus 2 (SARS-CoV-2) was declared as a pandemic by the World Health Organization (WHO) in March 2020 and has caused more than 164 million infections worldwide and approximately 3.4 million deaths, becoming a global health emergency [There has been no data to confirm that pregnant women may have a greater predisposition to infection; however, evidence suggests that SARS-CoV-2 infected pregnant women are at higher risk of hospitalization, admission to Intensive Care Unit (ICU), invasive mechanical ventilation (IMV) and extracorporeal membrane oxygenation (ECMO) therapy compared to the general population [Due to the physiological changes that occur during pregnancy, women are more vulnerable to respiratory infections and associated complications, with respiratory failure being a common cause of ICU admission , as the Although most of the coronavirus disease 2019 (COVID-19) cases in pregnant women are mild-moderate (88.5%), some women develop severe disease requiring oxygen therapy (9.8%), and up to 1.6% develop critical illness . CompareThere also seems to be an increased risk of ICU admission compared to non-pregnant women of reproductive age ,6, but tThe objective of the present study was to describe the characteristics of SARS-CoV-2 infected pregnant women admitted to the ICU and to investigate the causes and risk factors for ICU admission and whether obstetric pathology and type of delivery are relevant.This is a prospective multicenter study of consecutive cases of SARS-CoV-2 infection in a cohort of pregnant women registered by the Spanish Obstetric Emergency Group in 78 hospitals [A specific database was designed to record information regarding SARS-CoV-2 infection in pregnancy; the encoded data were entered by the principal researcher of each center during the enrolment period, which occurred at the time of the SARS-CoV-2 test during pregnancy and until 6 weeks after birth. We developed an analysis plan using recommended contemporary methods and followed existing guidelines for reporting our results  .We included all SARS-CoV-2 infected obstetric patients between 26 February and 5 November 2020 by testing suspicious cases admitted to the hospital due to compatible COVID-19 symptoms and by universal screening for SARS-CoV-2 infection, which was performed on all pregnant women on admission to the delivery ward (starting on 1 April 2020). SARS-CoV-2 infection was diagnosed by double-sample positive polymerase chain reaction (PCR) from nasopharyngeal swabs. All identified cases were included in the study, regardless of clinical signs and symptoms. Then, the infected patients were divided into two groups based on whether or not they required admission to intensive care units.All procedures were approved by the Drug Research and Clinical Research Ethics Committee of Puerta de Hierro University Hospital, Madrid, Spain, (Chairperson Prof. C. Avendaño Sola) on 23 March 2020 . Each collaborating center subsequently obtained protocol approval locally . The registry protocol is available on ClinicalTrials.gov (accessed on 1 November 2021), identifier: NCT04558996. Upon recruitment, mothers consented to participate in the study by either signing a document when possible, or by giving permission verbally, which was recorded in the patient’s chart in the electronic clinical recording system. Ethics committees approved the possibility of verbal consent during the first three months of the pandemic, given the contagiousness of the disease and the lack of personal protection equipment. Afterward, written consent (using a patient consent form) was collected from every patient who had previously given permission verbally.Information regarding the demographic characteristics, comorbidities and current obstetric history were extracted from the clinical and verbal history of the patient. Age and ethnicity were categorized following the classification used by the CDC (Centers for Disease Control and Prevention) , while dp-value was less than 0.05.For descriptive data, absolute and relative frequencies were used in the case of categorical variables and medians and interquartile ranges (IQR) in the case of quantitative variables. The possible association of maternal characteristics and perinatal events with ICU admission of patients were analyzed using the Pearson’s Chi-square test or Fisher’s exact test for categorical variables and the Mann–Whitney U test for quantitative variables . Statistical tests were two-sided and were performed with SPSS V.20 ; statistically significant associations were considered to exist when the To check the association of the perinatal complications that were statistically significant in the univariable analysis with ICU admission, the influence of confounding factors (and interactions) was controlled for with multivariable logistic regression modeling to derive adjusted odds ratios (aOR) with 95% confidence intervals (95% CI). Models were built for each perinatal complication separately, incorporating a range of variables and/or interactions after verifying the statistical association of potential confounding factors with ICU admission and the perinatal complication of interest  and in accordance with the ten-to-one event per variable rule to avoid model overfitting . ModelinDuring the study period, 1347 infected pregnant women were registered and analyzed in the 78 hospitals; 35 (2.6%) were admitted to the ICU, and 1312 (97.4%) did not require intensive care . Of pregThe answers to the following questions allow us to describe the results of our study.(p < 0.001). Furthermore, 71% of symptomatic patients admitted in the ICU developed pneumonia  as well as a higher proportion of women who had required in vitro fertilization  and a higher rate of multiple pregnancies . We did not observe any other differences between groups in relation to maternal characteristics, comorbidities or current obstetric history.Among our SARS-CoV-2 infected cohort, 51.1% of the patients were asymptomatic, and 48.9% had COVID-19 symptoms . The prop < 0.001), and its main reason was maternal respiratory worsening due to SARS-CoV-2 (51.4% of cases). We found that 77.4% (24/31) of C-sections performed in ICU patients were carried out before their admission to this unit and 22.6% (7/31) after they were already receiving intensive care.We observed that the cesarean section was much more frequent in the ICU group , while 40% were due to non-obstetric causes  . Additiop < 0.001, The proportion of women who developed pre-eclampsia was significantly higher in the group of patients admitted in the UCI  among ICU patients was nearly five times higher than in non-ICU patients  and to 106.2 (95% CI 17.4–7.7 × 108), respectively , both cases associated with Disseminated Intravascular Coagulation (DIC) . The first patient died in the operating room because of massive postpartum hemorrhages after abruptio placentae associated with DIC. The second patient died after several days in the ICU due to severe pneumonia with acute respiratory distress syndrome associated with septic shock and the development of DIC, which led to a massive bilateral PE and cardiac arrest.Maternal mortality in the entire series was 0.14%  and the considerable quantity of ICU admissions (35) included.The incidence of admission to the ICU in this series was 2.6%, lower than the one reported by previous studies . This diNo differences in maternal characteristics or comorbidities were observed between ICU and non-ICU patients, except for IVF and multiple pregnancies (higher rates among ICU). Prior to the COVID-19 pandemic, these conditions had already been described as risk factors for obstetric morbidity and, therefore, for ICU admission, due to a higher presence of comorbidities  and complications among IVF mothers ,14,15,16The high incidence of C-sections and preterm deliveries (mostly iatrogenic) that was observed in the ICU group could be explained by the urgency to terminate the pregnancy due to a worsening of the mother’s condition and respiratory failure, which was the most frequent complication described in ICU patients. We must always bear in mind that maternal oxygen consumption increases 20% during pregnancy owing to increased metabolic demands, and this, combined with a reduced functional residual pulmonary capacity, result in rapid desaturation during respiratory compromise . In one In our SARS-CoV-2 infected cohort, the main risk factor for ICU admission is the development of pneumonia. On the other hand, when pneumonia coexists with pre-eclampsia, the risk of requiring intensive care increases exponentially. Although SARS-CoV-2 infection primarily affects the respiratory system, its systemic manifestations, such as hypertension, renal disease, thrombocytopenia and liver damage, are also found and easily confused with pre-eclampsia . An incrThe incidence of hemorrhagic events in our entire series was 5.2% but increased up to 20% in those who required admission to the ICU. In previous studies, there was controversy over whether SARS-CoV-2 infection is a risk factor for postpartum hemorrhage ,26,27. IAnother consideration is the high rates of PE  ,29. ThisTherefore, surveillance and rapid intervention should be intensified in SARS-CoV-2 infected pregnant women with the mentioned risk factors and complications. Similarly, emphasis should be placed on anticoagulant therapy in these patients due to the increased thromboembolic risk, C-section surgery and immobilization in the ICU.As a limitation of this study, it should be highlighted that symptomatic patients are over-represented in our study population since not all participating hospitals had a universal antenatal screening program for SARS-CoV-2 infection (so only identified symptomatic cases) or implemented the program later. Another limitation is related to the identification and classification of pre-eclampsia cases, as angiogenic markers and echo-Doppler of the uterine arteries were not performed in most of the patients since this is not a common practice in many hospitals.n = 35) may have penalized the power of analyses, proven by the wide OR’s confidence intervals obtained in the multivariable analyses, which should be interpreted cautiously.Moreover, the small number of ICU admissions  could have led to an underestimation of the number of critical patients, as there were patients about to be admitted to the ICU but who were attended in these units. Additionally, the criteria for classifying the disease as mild, moderate or severe were not correctly implemented, and the specific medical treatment for COVID-19 was also very variable, depending on the protocols of each hospital and the moment of the pandemic, since they were frequently modified based on the emergent literature. Patients hospitalized for COVID-19 were treated with different drugs , and asymptomatic or symptomatic patients without hospitalization could receive or not prophylactic heparin.Respiratory failure due to SARS-CoV-2 pneumonia is the main cause of ICU admission among SARS-CoV-2 infected pregnant women. Multiple pregnancy or/and in vitro fertilization are risk factors for ICU admission, as well as pre-eclampsia and hemorrhagic events when they coexist with pneumonia. The higher incidence of C-sections and preterm deliveries among ICU patients are explained by the need to terminate the pregnancy due to a worsening of the mother’s condition.


Lymphovascular invasion (LVI) is an important prognostic indicator of lymph node metastasis and disease aggressiveness but clear molecular mechanisms mediating this in head and neck cancers (HNSC) remain undefined. To identify important microRNAs (miRNAs) in HNSC that associate with and are also predictive of increased risk of LVI, we used a combination of clustering algorithms, multiple regression analyses and machine learning approaches and analyzed miRNA expression profiles in the TCGA HNSC database. As the first step, we identified miRNAs with increased association with LVI as a binary variable. In order to determine whether the identified miRNAs would show functional clusters that are also indicative of increased risk for LVI, we carried out unsupervised as well as supervised clustering. Our results identified distinct clusters of miRNAs that are predictive of increased LVI. We further refined these findings using a Random forest approach, and miR-203a-3p, mir-10a-5p, and miR-194-5p to be most strongly associated with LVI. Pathway enrichment analysis showed these miRNAs targeted genes involved in Hippo signaling and fatty acid oxidation pathways that are mediators of lymph node metastasis. Specific association was also identified between the miRNAs associated with LVI and expression of several lymphangiogenic genes that could be critical for determination of therapeutic strategies. Recurrent or metastatic head and neck cancer (HNSC) that includes tumors of oral cavity, paranasal sinuses, nasal cavity, pharynx, and larynx is associated with poor patient outcome, tumor aggressiveness and is characterized by early metastasis to the regional lymph nodes . It is tLarge scale genome sequencing projects by The Cancer Genome Atlas (TCGA) have established extensive patient datasets of HNSC with detailed information of molecular characteristics of the tumor . As cancHence, in this paper, we address this critical gap and use sophisticated machine learning and clustering approaches to systematically identify specific clusters of miRNAs that are predictive of and show significant association with LVI and thus could be further evaluated as prognostic indicators of tumor spread in HNSCC. Further, a novel aspect of these studies was that as lymphangiogenesis or growth of new lymphatic vessels is a critical event that promotes LVI and subsequently lymph node metastasis (LNM), we also evaluated whether these miRNAs showed correlation with specific lymphangiogenic genes.Based on the 5% significance level criteria, we have selected 61 out of 496 miRNAs that are strongly associated with the LVI status. The odds ratio estimate and the 95% confidence interval for the association between LVI and the 61 miRNAs are given in For the purpose of illustration here we explain the odds ratio of the most significant miRNA, hsa-miR-203a-3p, in terms of the association between the miRNA and the LVI status. The result implies that the odds of having LVI decreases by 40% for one unit increase of the scaled hsa-miR-203a-3p. This is indicated in the side-by-side boxplot as shown in The pairwise correlation matrix of the 61 miRNAs reveals that hsa-miR-31-5p is highly correlated with hsa-miR-31-3p with a correlation of 0.96, hsa-miR-9-5p is highly correlated with hsa-miR-31-3p with a correlation of 0.91, hsa-miR-194-5p is highly correlated with hsa-miR-192-5p with a correlation of 0.90, and hsa-miR-30a-3p is highly correlated with hsa-miR-30c-2-3p with a correlation of 0.87. As this high correlation can cause interference to our next multiple regression analysis, we decided to remove hsa-miR-31-3p, hsa-miR-9-3p, hsa-miR-192-5p, and hsa-miR-30c-2-3p from the list of 61, resulting in 57 distinct miRNAs in the next step of analysis (data not shown).For identifying a predictive model for LVI, next we regressed LVI on the 57 miRNAs (after eliminating the 4 miRNAs that showed high correlation as described above). We used a multiple logistic regression method, and used the LASSO (Least Absolute Shrinkage and Selection Operator) approach to obtain regularized parameter estimates . The LASUsing the gap statistic, we found that the optimal number of clusters is three. For comparing the prediction power of each of the three supervised clusters, we fit a multiple logistic regression model for LVI on the miRNAs of each of the clusters separately. This resulted in three multiple logistic regression models and corresponding to each model we calculated the area under the ROC curve –5C. Our Further, an unsupervised hierarchical clustering of the 57 miRNAs were also represented as a heatmap along with the LVI status (Yes or No) . Both thFurther, to determine the most important miRNAs out of these 35 miRNAs that are identified by the Wilma cluster, we performed a random forest (RF) analysis as described earlier . In the Besides the association between the miRNAs and LVI, we also wanted to figure out the dependence among the miRNAs. For that we calculated partial correlation among the 35 miRNAs selected by the supervised clustering method. The partial correlation between two generic miRNAs, x and y, signifies the correlation between the two after eliminating out the effect of the rest 33 miRNAs. Thus, high partial correlations can be indicators of a possible causal link. It is important to note that partial correlation provides better evidence for regulatory genetic links than the pairwise correlation . Based oSince miRNAs directly regulate gene expressions by inhibiting the expression of specific mRNAs, in the next step we wanted to examine if there was any correlation between the 57 shortlisted miRNAs and some of the genes that play an important role in new lymphatic vessel formation or lymphangiogenesis  that couhttps://bio.tools/mirtarbase), and miRDB (http://www.mirdb.org/) [https://www.mirnet.ca) [The miRNAs in cluster 1 show highest association with LVI and are likely to regulate multiple genes and cellular pathways. Hence pathway analysis was carried out for the miRNAs in Wilma cluster 1 that showed highest association with LVI using the DIANA tools mirPath V3 . The miRdb.org/)  and the rnet.ca)  and the LVI has been established as an important indicator of disease outcome and treatment modalities in several cancers , 29. In It is important to note that metastasis is a complex process involving alterations in several cellular signaling pathways, dysregulation of multiple genes and involves a number of sequential steps not all of which are clearly defined. LVI is an important indicator of metastatic progression and in this study, our primary goal was to delineate the association between miRNA expression and LVI status and identify whether these important miRNAs grouped together in functional clusters that were associated with LVI. The supervised clustering technique is a novel way to achieve both goals. This method allows identification of groups of miRNAs that associate with LVI and likely regulate similar pathways thereby defining biologically relevant pathways that could be targeted to suppress LVI and thereby metastasis.In our analysis, the preprocessing step obtained by discarding lowly expressed miRNAs followed by step two pruning using the univariate logistic regression to reduce the noise as well as increase the power of detecting significant association. Thus, we obtain the probability of the presence of LVI for a new HNSC cancer patient based on the expression level of their preselected miRNA. Further downstream analysis involved supervised clustering to group miRNAs based on how the miRNAs are affecting the LVI status. The clustering function wilma makes use of the Wilcoxon sign-rank test statistic, adjusted by a well-defined margin function . It is cHence, for the purpose of analyzing the cellular pathways affected we focused on that cluster. According to the functional pathway analysis of the miRNAs in Wilma cluster 1, we found that the target genes of these miRNAs that were positively associated with LVI were associated with several important tumor enhancing processes, including TGF-β signaling pathway and ECM-receptor interaction mechanisms. This is significant as the TGF-β signaling pathway plays an important role in tumor metastasis and is involved in mediating early lymph node metastasis in multiple cancers , 47. TGFUsing RF approach, we identified several miRNAs that strongly correlated to LVI. Interestingly, almost all of the miRNAs that were identified by our random forest analysis to be significantly associated with LVI have also been independently identified in a number of studies in HNSC giving further credibility to our RF model. We focus our discussion here on the three miRNA  that were consistently identified by the multiple iterations of RF analysis and also belong to Wilma cluster 1 that shows highest predictive association with LVI. These miRNAs were chosen based on the fact that they were validated as the highest predictors across multiple analyses. We want to point out that our main three important miRNAs hsa-miR-203a-3p, hsa-mir-10a-5p, hsa-miR-194-5p fell in two different clusters based on the heatmap- hsa-mir-203-3p and hsa-mir-10a-5p in one cluster and hsa-mir-194-5p in other. This is not a surprise as the heatmap  did unsupervised clustering whereas Wilma provided supervised clustering. Secondly, the heatmap was based on the unscaled expression values whereas the k-means clustering and the Wilma clustering were applied on the scaled expression values. Our analysis showed that miR-203a-3p was negatively associated with LVI. This fits well with other studies where miR-203a-3p has been reported as a tumor suppressor and dysregulated in many malignancies including nasopharyngeal carcinoma , gastricTumor cells express high amounts of angiogenic molecules (that promote growth of new blood vessels) or lymphangiogenic molecules (that promote growth of new lymphatic vessels) that are critical for metastasis . HoweverIn conclusion, our results showed that specific miRNA clusters significantly associate with LVI. The identified miRNA clusters regulate multiple biological pathways that are involved in progression of LNM and could be potential predictors of metastatic disease. In addition, RF analysis revealed three miRNAs hsa-miR-203a-3p, hsa-mir-10a-5p, hsa-miR-194-5p to be most strongly associated with LVI and can be used as important prognostic indicators. Further, we also find that each of these miRNAs have association with significant molecular pathways as cell proliferation, metabolism and lymphangiogenesis that further promotes tumor progression. In addition, all of the identified miRNAs are associated with metabolic pathways as well as immune cell responses that define alterations in a metastatic lymph node so it also potentiates development of specific targeted interventions to these pathways that may distinguish a metastatic from a naïve lymph node. Different miRNAs regulate different aspects of metastatic progression and identification of specific miRNA linked with LVI, lymphangiogenesis and subsequent nodal metastasis may help in identification of patients at early stages of disease progression. Specific miRNA agonists (mimics) or antagonists (inhibitors) can be evaluated further in pre-clinical trials to evaluate their efficacy in inhibition of HNSCC progression when detected in earlier stages. Further studies are warranted to determine the functional relevance of these findings and evaluate the downstream targets in tumor models of HNSCC metastasis.https://xenabrowser.net/). We obtained mature miRNA sequences (IlluminaHiseq) referenced with a miMAT accession number. Particularly, the sequences are level-3 data, and log2, where total RPM represents the total of all isoform expressions for the same miRNA mature strand. The clinical data on subjects are also obtained through UCSC Xena, with the major focus on lymphovascular invasion status among many other phenotypic variables and in the downloaded data it was named as lymphovascular_invasion_present. This variable is referred to as LVI. Although the clinical data had information on 604 subjects, after removing subjects without any LVI information we were left with only 411 patients. Matching the miRNA sequence data with their corresponding clinical data, we obtained a total of 351 patients. The TCGA consortium contains data from five different sample types [https://www.bioconductor.org [We used the Head and Neck squamous cell carcinoma (HNSC) cohort of the TCGA data portal. The overall steps of the workflow are depicted in le types . Out of ctor.org . The HNSFor the statistical analyses, R software, version 3.6.2 was used. As a first step of pre-processing the data, we only considered miRNAs whose mean value across the patients were greater than one to avoid miRNAs with very low expression. After this exclusion, 496 miRNAs fit the criteria for further association studies. Finally, we standardized each miRNA expression to have zero mean and unit variance. Standardized values of every miRNA were obtained by subtracting the mean and then dividing by the standard deviation of the respective miRNA. The primary goal of our analysis was to identify the important miRNAs based on their association with LVI, or predictive power of the LVI status using statistical methods, and investigate their biological role in this context. At the very first stage, instead of working with all available miRNAs, we performed a second level of preprocessing to preselect a subset of miRNAs that are significantly associated with the binary LVI status based on the univariate logistic regression model. Specifically, we used the glm function to run the logistic regression. Based on the 5% significance level, we screened out important miRNAs that will be used for downstream analyses. Next, we computed the pairwise correlation among the miRNAs, and removed a handful of miRNAs that are highly correlated with others. Then, to find a best predictive model we fitted a multiple logistic regression model to LVI on all the miRNAs that were selected in the previous step. Since a large number of miRNAs are included simultaneously as predictors we used the penalized estimation method, the least absolute shrinkage and selection operator (LASSO) technique , to selek with their expected values under a null reference distribution of the data. The reference dataset is generated using Monte Carlo simulations of the sampling process. https://CRAN.R-project.org/package=pheatmap) [Besides finding a good predictive model, we wanted to examine if these miRNAs would show more connection with others and show functional clusters that are indicative of increased risk for LVI. Hence, clustering of miRNAs is a novel component of our analyses. Most of the supervised techniques rely on prior knowledge on the number of clusters . Therefoheatmap) .To determine the most important miRNAs from the set of miRNAs identified by the Wilma cluster, we performed a random forest (RF) analysis . To iden