Many milestones in medical history rest on animal modeling of human diseases. The SARS-CoV-2 pandemic has evoked a tremendous investigative effort primarily centered on clinical studies. However, several animal SARS-CoV-2/COVID-19 models have been developed and pre-clinical findings aimed at supporting clinical evidence rapidly emerge. In this review, we characterize the existing animal models exposing their relevance and limitations as well as outline their utility in COVID-19 drug and vaccine development. Concurrently, we summarize the status of clinical trial research and discuss the novel tactics utilized in the largest multi-center trials aiming to accelerate generation of reliable results that may subsequently shape COVID-19 clinical treatment practices. We also highlight areas of improvement for animal studies in order to elevate their translational utility. In pandemics, to optimize the use of strained resources in a short time-frame, optimizing and strengthening the synergy between the preclinical and clinical domains is pivotal. The accumulation of scientific publications eclipses the numbers generated during any other outbreak in the last 100 years; by March 2021 >110,000 COVID-19-related publications (PubMed) and >14,000 on preprint health science servers , e.g., compared to 114,124 all-time PubMed hits for “acute myocardial infarction”. The overall SARS-CoV-2/COVID-19 clinical information has outpaced data produced by animal-based studies . This is understandable given that animal studies aiming to decipher disease pathophysiology and testing of experimental therapies typically emerge as a secondary activity. However, a rapid expansion of preclinical testing capacity may markedly enhance anti-pandemic strategies, e.g. in a form of negative screening of potential drug candidates to exclude low-impact compounds and more efficiently streamline the drug and vaccine development processes. Notably, such an expansion contradicts long-term goals set by various inter policy makers (including the 2019 EU Directive (2010/63/EU) aiming at radical reduction and elimination of animal safety & drug testing 2https://opendata.ncats.nih.gov/covid19/animal).This chapter provides a focused overview of the animal modeling landscape in the specific context of the animal-to-human research interaction discussed in the subsequent chapters. Several recent reviews provide exhaustive species-specific modeling details ,6 and a Krt18 in epithelial lineage) Ace2 [Several species were already tested regarding their utility in reproducing COVID-19 . The Worge) Ace2 ,15. A trge) Ace2 ,17 or adge) Ace2 .Table 1OApart from ACE2, the newest evidence implicates additional receptors contributing to the SARS-CoV-2 tropism and cellular entry (e.g. acting as co-factors) with a potential propensity to specific tissues/cells. For example, neuropilin-1 and CD147 receptor were shown to be highly expressed in olfactory epithelial cells and brain ,19 whereLess experimentally prominent but infection-permissible  species include symptomatic (with pneumonia) minks ,22 and t2.1All WHO-recommended species  robustly shed virus via the respiratory tract and present with a varying magnitude of symptoms . Macaques, especially Macaca mulatta appears to be the most susceptible to SARS-CoV-2; they developed COVID-19-like lung lesions albeit with moderate symptoms NHP including cynomolgus macaques, African green monkeys and common marmosets recapitulate several features of human COVID-19 albeit with generally mild symptoms predominating (2.2While the majority of SARS-CoV-2 susceptible species display respiratory symptoms (including COVID-19-like pneumonia), they generally lack hypoxemic respiratory failure, extrapulmonary organ dysfunction and lethality . Two rec3Animal COVID-19 models should ideally provide a full recapitulation of COVID-19 pathophysiology represented by an array of the clinical phenotypes and their sequelae and be widely available. Current high demand for experimental animals exceeds capacity of commercial breeders impeding the investigative progress and verification of the existing findings. The limited lethality across SARS-CoV-2/COVID-19 models constitutes a critical shortcoming , Table 2Other shortcomings are difficult and impossible to overcome. Immuno-modulatory agents against COVID-19 are difficult to investigate given different inter-species immune responses. To improve mechanistic understanding, a given therapeutic should preferably be tested in different species. Furthermore, while the availability and ease of use of experimental animals are inversely correlated to their size, the physiological and anatomical similarities are not. For example, the lethal phenotype and neurological sequelae position the mouse as a translationally-attractive model for COVID-19. Unfortunately, the mouse is an inferior model of human airway diseases given its e.g., monopodial (versus human trichotomous) airway branching system, much smaller alveolar and ventilatory unit diameter, richer bronchus-associated lymphoid tissue and differences in regenerative pulmonary response 4in-vivo testing. The first study testing a traditional Chinese medicine in hACE2 mice was published on May 5, 2020 The emergence of the first reproducible models that partly recapitulate COVID-19 has enabled limited 5https://www.who.int/publications/m/item/draft-landscape-of-covid-19-candidate-vaccines). Twenty vaccines entered phase 3 trials and demonstrated interim benefits have led to regulatory approval/authorization of 10 vaccines https://www.nytimes.com/interactive/2020/science/coronavirus-vaccine-tracker.html). The development process of COVID-19 vaccines, including some of the most advanced Janssen-Cilag In February 2021, the WHO reported 63 candidate vaccines in clinical trials and 179 in preclinical evaluation , the increase of clinical studies since the onset of the pandemic is unprecedented. As of March 2021, there were over 6400 entries on COVID-19 clinical studies. The following section provides a summary of the currently active/recruiting phase II-IV trials testing immuno-modulating substances, which subjectively constitute: i) the largest and most dynamic therapeutic domain, and ii) the most challenging area for animal-to-patient translation and iii) it remains unclear whether hypo- or hyperinflammation responses dominate in COVID-19 progression. The subsequent sections discuss key challenges related to the design and execution of clinical trials.According to registry data  Four randomized clinical trials  indicated a potential clinical benefit of intravenous dexamethasone/hydrocortisone in critical COVID-19 patients 6.2Multiple trials evaluate modulation of diverse cytokines: either as application of the recombinant protein, such as granulocyte-macrophage colony-stimulating factor , interferon (IFN)-α , the neutralization of cytokines such as macrophage colony-stimulating factor , interleukin (IL)-6 , IL-1β , tumor necrosis factor (TNF) , TNF superfamily member 14 , and their receptors such as GM-CSF receptor , IL-1Ra , and IL-6R . Few studies target chemokines e.g. IL-8  or chemokine receptors, such as CCR5 . The majority of these trials use humanized antibodies and few trials employ small molecule-based inhibitors.www.recoverytrial.net). Similarly, the off-label use of recombinant human IL-1Ra (anakinra) in 52 COVID-19 patients was associated with reduced ICU admission for mechanical ventilation and death Immunotherapies directed at pro-inflammatory cytokines were suggested as adjunctive treatment in COVID-19. While smaller studies provided variable results with anti-IL-6 (siltuximab) and anti-IL-6 receptor (tocilizumab) antibodies 6.3Several trials target downstream signaling cascades of receptors of cytokines, chemokines and pathogen recognition receptors: 24 trials test the inhibition of the JAK pathway (Ruxolitinib), six trials of inhibitors of the Bruton's tyrosine kinase, two trials of sphingosine kinase 2 (Opaganib) and one trial inhibition of phosphoinositide 3-kinase. Two studies target pattern recognition receptors toll like receptors (TLRs) 2/6/9 and cytosolic NLRP3, the receptor component of the canonical inflammasome complex. Regarding T-cells, divergent approaches aim at either boosting the cellular activity with Thymosin-1α  and anti-program cell death protein (PD)-1 antibody  and inhibiting its activation with cyclosporine  and IMU-838 . 71 trials evaluate the application of recombinant interferons, alone or in combination with other drugs, with IFN-α  and β  to boost the T cell activity. Summarizing, based on the emerging positive results in patients with a milder COVID-19 ,90 and e6.4Cell based therapies such as intravenously and intrapulmonarily delivered mesenchymal stem cells (MSC) have gained interest as a potential treatment in COVID-19-related lung injury given their anti-inflammatory and immunoregulatory abilities with possible effects on the lung tissue repair. Presently, three published phase I trials investigating MSCs in severe COVID-19 showed promise Overall, 55 clinical trials investigating cell-based therapies are currently underway , using MSCs of different origins  and isolated SARS-CoV-2 specific T cells. Furthermore, native natural killer (NK) cells  and genetically modified NKG2D-ACE2 CAR-NK cells  are tested; both strategies aim at recognizing/inactivating virus-infected ACE2-expressing cells. To increase efficacy and reduce the cytokine release syndrome (CRS) risk, those genetically engineered NK-cells are modified to secrete both GM-CSF neutralizing single-chain variable fragment (scFv) and an IL-15 agonist (sIL-15/IL-15Rɑ chimeric protein) inhibiting the otherwise short NK-cell survival time. Summarizing, there is no evidence currently supporting the use of cell-based therapies in COVID-19. To evaluate survival and tissue repair effects of cell-based therapies in COVID-19, randomized phase 2/3 trials with a long-term follow up and relevant mechanism-of-action endpoints are required.6.5The use of nonsteroidal anti-inflammatory drugs in COVID-19 patients is deliberated 6.6In animals, there has been some evidence that neutralizing antibodies (NAbs) are protective against SARS-CoV-2 infection generating an interest in their potential prophylactic/therapeutic use against COVID-19. The available NAbs primarily block the glycoprotein that mediates binding to the RBD of the host ACE2 (RBD) 7https://www.remapcap.org) . To at least partly remedy such challenges, the WHO advised developing “Master Protocols”, especially for high-powered, multi-center trials to support the generation of high-quality data (https://www.who.int/blueprint/priority-diseases/key-action/novel-coronavirus/en/).Previous pandemics outlined the urgent need for evidence to guide the best treatments. However, pandemics should not simultaneously serve as an excuse for lowering scientific standards One of the key complexities is that the precise nature of the infecting pathogen(s), clinical consequences and suitable interventions (particularly pathogen-specific) are not known in advance and inferring information from previous pandemics of related origins can be misleading and dangerous 7.1ad hoc availability with a trade-off of data/analytical quality A typical pandemic elicits a high number of cases occurring over a short period . In the early phase of the COVID-19 pandemic, researchers heavily relied on so-called “real world” data e.g. from sources such as electronic medical records, patient registries, reimbursement/claims data or published single center case series. All of these have an advantage of an vs. Bayesian statistics are of lesser importance compared to these points When more traditional clinical trials are considered, one or few research questions are typically the driver of a prospectively designed study with a study protocol. The pandemic has taught us that such trials are possible and lead to valid conclusions. However, we have also learned that they have to be pragmatic - often large and simple - and only work with an established technical and organizational infrastructure. The latter includes appropriate responses from ethical committees and the public as well as hospital authorities. The often cited major difference between trials that rely on Frequentist 8Design of animal research for translational purposes should be largely aimed at supporting the preparation and execution of clinical trials. This should be done by using the entire available pre-clinical armamentarium including pathomechanistic investigations, preselection of therapeutics, identification of non-and responder subgroups, identification and optimization of endpoints. To achieve desired synergy, such a process requires knowledge on the limitations and advantages of a given model and intricacies of proper study design that maximally synchronizes with the stringent demands of the clinical-level research. Especially in a pandemic, such converging areas should be developed given the pressing need for a rapid understanding of COVID-19 and improvement of patient outcomes. Below, we present investigative areas with the strongest potential for enhancement of the animal-to-human investigative synergy.8.1Both mild and severe COVID-19 phenotypes are instrumental for adequate pre-clinical testing of preventive and therapeutic strategies. The currently predominating mild-to-moderate model phenotype should not be reflexively rejected as inadequate, however, given that it enables detection of any adverse effects of tested substances. By employing high lethality of given disease models, a frequent design feature in critical illnesses, such a design is primarily set toward observing hypothesized benefits rather than undesired complications ,117. In 8.2Compared to human trials, animal studies have a strong advantage regarding pre-selection features. Comparative testing can be relatively easily executed in divergent homogeneous cohorts displaying a desired pre-defined characteristic, e.g. male/female, young/old, early/late, lung/gastro-intestinal phenotype. In trials, these elements are typically assessed retrospectively (thus with weaker cause-effect relationship powers). Some treatments may elicit contrasting effects depending on the COVID-19 characteristics and/or type of targeted patients; a defined pre-clinical cohort-testing will likely provide an informational groundwork for clinical investigations. Animal cohort studies can be i) performed before a trial is launched, ii) run parallel to an ongoing trial to support testing of defined hypotheses  and/or iii) rapidly executed to confirm/refute observations from ongoing and finished trials. The emerging evidence underlines the importance of cohort-oriented research; aged patients are at risk for unfavorable outcomes whereas females are less prone to the most severe COVID-19 phenotypes 8.3Relevant animal models, either wild-type or genetically manipulated, appear indispensable for investigation of specific SARS-CoV-2-induced pathomechanisms such as hypercoagulability and immuno-inflammatory responses  associated with various disease phenotypes. The explorative possibilities should not be limited to molecular research but also assess physiological responses  are capable of overcoming them. Such impediments delay any developmental process and limit possible options.Priority 2: Promotion of expert networks and data sharing. It appears beneficial to promote establishment of pre-clinical networks of expert-consortia with resources enabling a rapid launch of research whenever required. Such consortia should combine academic, non-profit and for-profit research centers and be authorized to rapidly and consensually share information. A formalized collaborative format will exploit available synergies and lead to development of rigorous and reproducible animal models, which can be then quickly implemented for investigative purposes.Priority 3: Safeguarding stable funding and ethical approval. Given the fact that existing expert consortia may best allow for timely launch of animal model studies, a consensus public funding and ethical approval (across the entire spectrum of severity) should be rapidly obtainable for respective expert-institutions. We propose that the respective national government funding agencies and ethical committees have consensually prepared contingency plans allowing a rapid and synchronized launch of the designated pre-clinical consortia.9.2While multiple potential biological targets may be known (or inferred), classic drug development is delayed in a fluctuating pandemic and the presence of virulent pathogens. A global medical crisis limits implementation of state-of-the-art designs and not all available drug candidates can be simultaneously tested.. It is most efficient to use existing, approved substances whose efficacy, risks are known from other indications, and from which a therapeutic benefit in COVID-19 (or any future emergency disease) can be assumed. Experimental substances may potentially offer excellent treatment benefits but should be viewed as secondary testing choices due to a protracted safety/efficacy testing process and high risk of failure.Priority 1. Judicious selection of therapeutics for initial testingPriority 2. Reliance on high-quality multicenter pragmatic RCTs. Most of the available SARS-CoV-2/COVID-19 evidence stems from low-quality studies. This diverse non-RCT data segment, although attractive given its simple design and relatively rapid data generation, typically provides unreliable findings that may provoke erroneous changes in treatment guidelines. High-quality RCTs should be preferred given that despite delay, they provide the most reliable evidence of treatment efficacy, based on which lasting treatment recommendations can be formed.Priority 3: Separation of patients based on their phenotypic characteristics. COVID-19 has multifaceted presentation and accurate characterization of various disease phenotypes is central for selection of adequate therapies (e.g. with immunomodulators). Such a cohort selection allows i) precise trial enrollment of defined patient types and ii) reverse-translation of the specific subgroups into respective animal cohorts for further characterization and testing of therapeutics. Previous trial failures (e.g. in sepsis) imply that such phenotypic separation is pivotal for the development of individualized interventions.101How to most effectively facilitate  and streamline the development of animal models so they achieve a maximal fidelity to the clinical COVID-19 presentations?2Which elements of the already known evidence should be prioritized for bidirectional verification between animal and clinical studies to enhance understanding of COVID-19 pathophysiology?3How to organize, coordinate and optimize global research-exchange networks amalgamating both pre-clinical and clinical evidence in a pandemic?4How to effectively promote and facilitate preference for advanced multi-center RCTs over proliferating low-quality trials?Using all available tools, scientists and physicians share an accountability to understand pathophysiology of the diseases they combat to maximize the efficacy of treatments and minimize adverse effects. In the translational research context, the current pandemic provokes several questions aimed at operationalization of the above-formulated priorities:11Testing of emerging SARS-CoV-2 mutations section).First, panels of 2–3 authors, selected based on their expertise, created individual review chapters/sections/tables. Second, each draft was critically reviewed by all other authors. Third, all authors revised their drafts based on the received feedback and carried out a final update to include all most recent relevant studies published during the writing of the manuscript. The first and corresponding authors conducted the final assembly of the reviewed chapters. To identify the relevant information, the authors searched PubMed and/or Medline for articles published in English by December 2020, using a panel of search terms and/or combinations thereof, always including one of the following terms: "SARS-CoV-2", "COVID-19", "MERS-CoV", “SARS-CoV”, "SARS-CoV-1", "coronavirus". Search of medRxiv/bioRxiv was performed to identify and monitor progress of potentially relevant publications but referencing to publications without peer-review was kept to minimum and only when necessary (e.g. in the 12TS, JMC, MK, SW, GL, IML, SC, FU and MFO wrote the Animal Modeling-related sections; FMB, EJGB, SBF, MGN, MG, AS, SC, FV, RF and AGS wrote the Clinical Trials-related sections; IR, TS, MSW and MFO wrote the Animal-Human Interactions section; MFO, MSW, TS, AT, JCS and IR wrote Introduction, Synthesis and Outstanding Questions. Additionally, IR, MW and MFO created The authors declare no conflicts of interest.


Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve in humans. Spike protein mutations increase transmission and potentially evade antibodies raised against the original sequence used in current vaccines. Our evaluation of serum neutralizing activity in both persons soon after SARS-CoV-2 infection (in April 2020 or earlier) or vaccination without prior infection confirmed that common spike mutations can reduce antibody antiviral activity. However, when the persons with prior infection were subsequently vaccinated, their antibodies attained an apparent biologic ceiling of neutralizing potency against all tested variants, equivalent to the original spike sequence. These findings indicate that additional antigenic exposure further improves antibody efficacy against variants. Beta (Gamma (Evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein results from selection for random mutations that yield fitness benefit that promotes person-to-person dissemination of the virus. Mutations in the receptor-binding domain (RBD), which is involved in viral entry and the key target of neutralizing antibodies, may also facilitate viral resistance to antibodies against the original spike sequence either from prior infection or the current vaccines. Spike variants such as B.1.351/a  in April 2020 or earlier, of vaccinated persons who had no prior history of infection, and of the persons who recovered from COVID-19 after subsequent vaccination. This examination directly compares serum neutralization of seven commonly observed spike variant combinations of five RBD mutations by antibodies elicited by vaccination alone, SARS-CoV-2 infection alone, and combined infection and vaccination, to evaluate whether repeated antigenic exposures would increase efficacy of humoral immunity to cope with spike mutations.Beta , potency against spike variants was variably less than against the original sequence contained in the vaccine . The mutMutations in RBD also variably reduce the spike-neutralizing activity of antibodies elicited by natural infection with a similar pattern. Serum antibody spike-neutralizing potency was also assessed for 10 persons with recent mild SARS-CoV-2 infection , raw anti-RBD antibody measurements, and raw neutralization titers. Download Copyright © 2021 Ibarrondo et al.2021Ibarrondo et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Vaccination of previously SARS-CoV-2-infected persons increases neutralizing potency against variants to an apparent biologic ceiling. When the prior-infected individuals were vaccinated after approximately a year , serum neutralizing potency against the original sequence spike was relatively unchanged, but activity against all variants equalized to a level similar to the original sequence . NeutralBeta  potency against the original sequence was already maximal, and (ii) the additional antigenic exposure from vaccination further increased antibody potency against suboptimally recognized variants to reach the same maximal potency.This gained ability is compatible with further maturation of humoral immunity after SARS-CoV-2 infection and vaccination, consistent with reports of ongoing affinity maturation after resolved infection –9. It is–Overall, our findings raise the possibility that resistance of SARS-CoV-2 spike variants to antibodies can be overcome by driving further maturation through continued antigenic exposure by vaccination, even if the vaccine does not deliver variant sequences. Whether this can also be accomplished in SARS-CoV-2-naive persons through vaccination alone, such as delivering supplemental doses beyond the original vaccination regimen of two doses, remains to be determined.Volunteers provided informed consent under an institutional review board-approved protocol. Persons with mild SARS-CoV-2 infection were evaluated shortly after recovery from illness, and these persons as well as healthy control individuals without prior SARS-CoV-2 infection were evaluated soon after vaccination for COVID-19. The persons with prior infection all had symptom onset no later than April 2020 in the United States, when the SARS-CoV-2 variants were not yet reported to circulate, and all had mild infection that did not require hospitalization  as recently described in detail , 15. In 16–80). The “neutralizing potency”  was defined as the ratio of neutralization activity (DF80) to anti-RBD antibody concentration , adjusting neutralizing activity for antibody quantity. All data are given in Spike neutralization activity was assessed with a spike-pseudotyped lentiviral vector assay as recently described in detail . Briefly


To describe a case of complete spontaneous regression of a middle ear melanoma.We present a case of a 68-year-old man with complaints of unilateral hearing loss and an ipsilateral facial nerve paresis. Radiological and histopathological examination revealed a cT4bN0M0 mucosal melanoma of the middle ear.The patient underwent a subtotal petrosectomy and postoperative radiotherapy.Computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography/computed tomography with 2-[fluorine-18]-fluoro-2-deoxy-D-glucose (FDG-PET-CT), and histopathological examination.After subtotal petrosectomy, histopathological examination of the resection specimen showed only fibrosis and a histiocytic and clonal T-cell infiltration, but no residual melanoma at the primary tumor site, consistent with spontaneous tumor regression. Follow-up MRI scanning 6 and 12 months after radiotherapy showed no signs of tumor recurrence.This case describes the concept of spontaneous regression of a mucosal melanoma of the middle ear. Spontaneous tumor regression at this location has not been described before. A 68-year-old male patient was referred to the department of otorhinolaryngology because of left-sided, progressive hearing loss. He was suspected of having an otitis media with effusion. However, routine clinical examination after 3 months showed an atypical granulomatous presentation of the tympanic membrane and therefore a biopsy was taken. Histopathological examination showed a highly atypical and mitotically active stromal melanocytic proliferation, positive for S100, Melan-A, and MITF  was almost in remission. The patient had no complaints of otalgia or otorrhea and there was no history of an ear trauma. He had a history of hypertension, atrial fibrillation, coronary artery disease, depression, and obstructive sleep apnea syndrome. There was no family history of melanoma. At physical examination, inspection of the facial musculature showed a mild degree of asymmetry of lip closure (HB II/VI). Otomicroscopy showed a mass at the level of a seemingly thickened (or absent) tympanic membrane with blue discoloration at the posterior upper and lower quadrant post-biopsy. This mass was non-pulsatile and the usual anatomical landmarks such as the annulus fibrosus tympanicus and the tympanic membrane could not be identified. The Rinne test using a 512-Hz tuning fork was negative on the left side and the Weber test was lateralized to the left ear. Further physical examination including nasal endoscopy and palpation of the neck showed no abnormalities. The audiometry examination showed a left-sided mixed hearing loss of 92 dB HL with maximum speech recognition of 88% at 110 dB SPL.Computed tomography (CT) and magnetic resonance imaging (MRI) revealed soft tissue invasion of the left mastoid bone and the middle ear cavity, completely embedding the ossicular chain Fig. . DehiscePositron emission tomography/computed tomography with 2-[fluorine-18]-fluoro-2-deoxy-D-glucose (FDG-PET-CT) demonstrated intense FDG-uptake at the left middle ear cavity indicative for a malignant tumor based on the histopathological diagnosis Fig. A. Only mThe tumor was staged according to the 8th TNM Classification for Mucosal Melanoma of the Head and Neck as a cT4bN0M0 malignant mucosal melanoma originating from the left middle ear cavity .One month after presentation at our outpatient clinic, a subtotal petrosectomy was performed. During surgery, suspicious brown-colored granulation tissue was obtained from the middle ear cavity and mastoid bone. Furthermore, several defects of the tegmen tympani were present at the level of the middle and posterior cranial fossa. The granulation tissue was removed from the mesotympanum, hypotympanum, sinus tympani, oval window, antrum, mastoid cavity, and perilabyrinthine cell tracts. Microscopically radical resection of the aberrant tissue was not possible due to the extensive spread. The middle ear cleft was obliterated with abdominal fat.+, partly CD4+, partly CD8+ T-cell infiltrate, and CD68+ histiocytic infiltrate was observed within a large area of fibrosis and dilated blood vessels. Additional melanocytic stainings  were all negative  and a FDG-PET-CT follow-up scan revealed minimal soft tissue induration at the petrosectomy site and mild FDG uptake with no evidence of residual tumor Fig. B. Since Malignant melanoma develops through the neoplastic transformation of pigment-producing cells, usually located in the epidermis and dermis . Accumul+ and CD8+ T-cells directed against melanoma cells  performed on tissue from this area showed a highly clonal T-cell response, further supporting the phenomenon of regression  have been reported in the literature ,10,14. O


V-D-J genes. Somatic hypermutation (SHM) of immunoglobulin heavy chain variable (IGHV) genes are used as a prognostic marker in B-cell malignancies. Clinically, IGHV mutation status is determined by targeted Sanger sequencing which is a resource-intensive and low-throughput procedure. Here, we describe a bioinformatic pipeline, CRIS (Complete Reconstruction of Immunoglobulin IGHV-D-J Sequences) that uses RNA sequencing (RNA-seq) datasets to reconstruct IGHV-D-J sequences and determine IGHV SHM status.B cells display remarkable diversity in producing B-cell receptors through recombination of immunoglobulin (Ig) IGHV gene sequence. CRIS improves on existing tools that infer the B-cell receptor repertoire from RNA-seq data using a portion IGHV gene segment by de novo assembly. We show that the SHM status identified by CRIS using the entire IGHV gene segment is highly concordant with clinical classification in three independent chronic lymphocytic leukemia patient cohorts.CRIS extracts RNA-seq reads aligned to Ig gene loci, performs assembly of Ig transcripts and aligns the resulting contigs to reference Ig sequences to enumerate and classify SHMs in the https://github.com/Rashedul/CRIS.The CRIS pipeline is available under the MIT License from Bioinformatics Advances online. AdditioIGHV) genes are prognostic markers in different B-cell malignancies, including chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL) and follicular lymphoma   were designed to reconstruct only the CDR3 region, representing only a portion of the IGHV gene, while the entire IGHV gene segment is required to determine the SHM status in B-cell malignancies. In addition, these tools have not been validated against gold standard PCR-Sanger datasets for SHM classification. To address these gaps in determining IGHV mutational status in B-cell malignancies, we developed a bioinformatic pipeline, CRIS (Complete Reconstruction of Immunoglobulin IGHV-D-J Sequences), which extracts RNA-seq reads aligned to putative Ig loci, assembles the complete IGHV gene, identifies the most abundant Ig transcript and enumerates SHMs by comparison with germline reference sequences. Classification of IGHV mutational subtypes by CRIS was validated against PCR-Sanger-based clinical classification in three independent cohorts of CLL patients and shown to be comparable.Several bioinformatic pipelines have been developed to infer BCR repertoire from RNA-seq data, including ABRA (n = 16) and used according to procedures approved by the Research Ethics Board (REB H12-01767) of the University of British Columbia (In the Centre for Epigenomic Technology (CEMT) cohort, peripheral blood samples were obtained from CLL patients undergoing treatment at BC Cancer (Columbia . RNA washttp://www.ihec-epigenomes.org). These guidelines as well as the standard operating procedures for RNA-seq library construction and sequencing are available at https://thisisepigenetics.ca/for-scientists/protocols-and-standards and by request. Additional CLL patient RNA-seq datasets with matching IGHV mutation status were collected from published datasets: GSE66228  with the germline IGHV sequences, we used blastn   and MiXCR (v3.0.3) were run on the RNA-seq bam file generated by STAR (v2.7.5a) aligner  for Trinity to assemble the Ig transcript. However, as expected, the overall number of Ig transcripts identified correlated with the sequencing depth. Based on this analysis, we developed a pipeline called CRIS and benchmarked its ability to call IGHV mutation status. In the CRIS pipeline, we automate the process of read extraction from our novel putative Ig coordinates, perform quality trimming of selected sequence reads, assemble transcripts, enumerate transcript abundances and identify somatic mutations using reference germline sequences for SHM classification.Having established that enrichment of sequences using our Ig feature set significantly reduced compute resources without a reduction in the sensitivity, we next examined the impact of RNA-seq sequencing depth. For this, we leveraged a set of 16 CLL RNA-seq libraries with an average of ∼28 million paired reads from GSE66228  is clinically defined by IGHV sequence alignments of >98% identity to the reference sequence  that did not change the SHM classification. In seven of the eight divergent cases, the percent IGHV identity reported by the Sanger-based test was higher compared to CRIS  spanning the CDR3 region. Thus, on average, V’DJer assembled 75.44% of the IGHV gene whereas CRIS reconstructed 99.74%  compared to V’DJer . Using deeper RNA-seq datasets (∼300 million reads) V’DJer took five times more time to run than CRIS . Using 16 threads, TRUST took 36 wall-clock minutes on average using GSE66228, an order of magnitude longer than CRIS. MiXCR (IGHV mutation status in CLL at a sensitivity and specificity equivalent to current Sanger-based clinical tests. Furthermore, CRIS was able to reconstruct the entire IGHV sequence thus increasing the accuracy of SHM classification. This is in contrast to a majority of existing pipelines designed to infer only CDR3-derived sequences (PCR-Sanger-based Ig SHM classification is resource-intensive, subject to PCR bias, and suffers from an ∼9% to 18% failure rate (IGHV mutations (IGHV genes is either not reported or partially reported. Furthermore, for a majority of publicly available RNA-seq datasets where SHM status is reported, detailed IGHV mutation reports with gene name, percent identity and clonal frequency are not available restricting the ability to assess mutational values. To meet this need, we developed CRIS and demonstrated its ability to rapidly classify IGVH mutational status with clinical accuracy. We anticipate that CRIS will prove to be useful in the mining of available B-cell RNA-seq datasets and that it will provide a framework to incorporate RNA-seq as a diagnostic tool to examine the BCR clonal rearrangement and SHM status.A registry of ∼1500 CLL patients showed that 90% of patients were not screened for vbab021_Supplementary_DataClick here for additional data file.


The human body’s ten trillion cells are constantly assailed with environmental insults and genetic susceptibilities that can initiate tumor formation. Yet, most people live cancer-free for decades. This bewildering feat is due, in part, to the remarkable ability of our immune system to recognize and eliminate tumor cells. Unfortunately, 12,000 Americans/year are diagnosed with a rare, aggressive, and fatal tumor that escapes immune recognition: glioblastoma. Here, we continue efforts to develop a treatment capable of stimulating immune recognition of glioblastoma. The treatment is based on an oncolytic virus that causes tumor-selective infections. Neural stem cells are used to enhance viral distribution throughout the tumor. This study selects a dosing strategy to enable more comprehensive viral inoculation of the tumor than was possible in our previous clinical trial. This research demonstrates to the broader community that multiple-cycle oncolytic virotherapy may be therapeutically beneficial despite an anti-viral response after the first administration.11 viral particles). Higher doses were not assessed due to volume limitations for intracerebral administration and the inability to further concentrate the study agent. It is possible that therapeutic efficacy could be maximized by administering even higher doses. Here, we report IND-enabling studies in which an improvement in treatment efficacy is achieved in immunocompetent mice by administering multiple treatment cycles intracerebrally. The results imply that pre-existing immunity does not preclude therapeutic benefits attainable by administering multiple rounds of an oncolytic adenovirus directly into the brain.Tumor tropic neural stem cells (NSCs) can improve the anti-tumor efficacy of oncovirotherapy agents by protecting them from rapid clearance by the immune system and delivering them to multiple distant tumor sites. We recently completed a first-in-human trial assessing the safety of a single intracerebral dose of NSC-delivered CRAd-Survivin-pk7 (NSC.CRAd-S-pk7) combined with radiation and chemotherapy in newly diagnosed high-grade glioma patients. The maximum feasible dose was determined to be 150 million NSC.CRAd-Sp-k7 (1.875 × 10 Oncolytic virotherapy is a promising approach for treating drug- or radiation-refractory brain cancer. Oncolytic viruses (OV) kill tumor cells directly via oncolysis; and also indirectly, by stimulating anti-tumor immune responses. Among oncolytic viral species used in clinical trials, adenoviruses possess several advantageous properties, including relatively simple genetic modification, inherent immunogenicity, and high viral titer production . To dateNeural stem cells (NSCs) have the intrinsic capability to migrate to invasive primary and secondary brain tumor sites in various preclinical models , whether11 Viral Particles) . It. It22]. Increasing the administered volume is not a possibility, either through intratumoral injections or intraventricular, due to safety concerns . We therThe ability of complement proteins in serum to neutralize free CRAd-S-pk7 independent of pre-existing antibody presence is demonstrated both in vitro and in vivo . We condWe also performed an in vivo experiment in immunocompetent mice in which CRAd-S-pk7 presence was visualized 1 day after administering either one, two, or three rounds of intra-tumoral treatment with either free CRAd-S-pk7 or NSC-CRAd-S-pk7. CRAd-S-pk7 transduction of GL261 glioma cells was prevented even with the first round of treatment, before the naive mouse had anti-Ad5 anti-bodies present. Furthermore, NSC-mediated CRAd-S-pk7 delivery enabled intra-tumoral CRAd-S-pk7 to be present across all three rounds of administration B.5 NSC.CRAd-S-pk7s  in a more challenging, larger tumor model . In the powered experiment, no survival benefit was seen with a single administration of NSC-CRAd-S-pk7. The median survival was 35 days in the group receiving a single dose of NSC. CRAd-S-pk7, while the mice treated with three weekly rounds was extended to 41 days  , suggesting no obvious adverse effects of repeated NSC-CRAd-S-pk7 treatment  for multiple rounds of NSC-CRAd-S-pk7 were performed by monitoring liver and kidney enzymes using GLP standards. There was no observable difference between any of the groups  .F4/80 staining demonstrates that tumors appear noticeably smaller after one round of NSC.CRAd-S-pk7 treatment when compared to the sham control group E. The brNovel treatments that can improve survival for glioblastoma patients are urgently needed. NSC.CRAd-S-pk7 is a novel therapeutic product under clinical investigation, but its true therapeutic potential cannot be adequately tested until the dose-limiting volume restrictions are resolved. The results presented here motivated our decision to pursue intracerebral administration of multiple weekly doses. This approach will be clinically tested for safety in an upcoming Phase 1 trial (IND 19532).The most important result of these IND-enabling studies is that three weekly intracerebral rounds of a clinical-equivalent NSC.CRAd-S-pk7 dose improved the survival of brain tumor-bearing immunocompetent mice relative to a single administration. Possible mechanisms that may explain the superior efficacy of three administrations are: (1) a higher viral load more effectively competes with replicating tumor cells; (2) temporally spaced intra-tumoral injections improve NSC.CRAd-S-pk7 distribution and enable virus deposition in different and noncontiguous tumor regions ,25,26; aEven though our result is one of only a few studies demonstrating the superiority of repeat dosing versus single-cycle OV therapy, this idea is consistent with a growing body of work, indicating that pre-existing immunity does not eliminate the therapeutic efficacy of oncolytic virotherapies, particularly with intra-tumoral administration. In fact, preclinical studies show increased effectiveness due to the anti-viral immune response against oncolytic virotherapy for Newcastle disease virus, maraba, and reovirus, although the mechanisms are unknown ,28,29. SWhen reflecting on the clinical tumor regressions in select brain tumor patients, it is worth noting that these regressions occurred months after OV administration, when no active viral replication was detectable. Furthermore, the tumors initially increased in size, similar to the pseudo-progression observed after other immunotherapy treatments . Lastly,Our study differs from others in that we are using a cell delivery vehicle that is not necessarily inert with respect to how the immune system recognizes and clears the virus, or how it interacts with the host immune system. Our NanoString transcriptome analysis suggests that, in general, the immune changes that occur in response to NSC.CRAd-S-pk7 treatment seem to be driven by the virus itself, as only minor fluctuation is seen when delivered via NSC. This finding is consistent with our previous findings that NSCs are generally immune-privileged, characterized by lack of major histocompatibility complex class 2 (MHC-II) expression and low MHC class 1 (MHC-I) expression , and tho11 Viral Particles). In a study of one vs. six cycles of measles virotherapy for ovarian cancer [Our immediate translational objective restricted our study to a dose-equivalent to 150 million NSC.CRAd-S-pk7s  attract further immune surveillance to the otherwise cold tumor microenvironment and (2) alert the immune system with updated information regarding tumor adaptations. Future work will focus on maximizing activation of anti-tumor immunity, perhaps through co-administration with checkpoint inhibitors or rapamycin, or in conjunction with anti-GBM CAR-T/NK cell treatments.


Camptothecin (CPT), a natural plant alkaloid, has indicated potent antitumor activities via targeting intracellular topoisomerase I. The promise that CPT holds in therapies is restricted through factors that include lactone ring instability and water insolubility, which limits the drug oral solubility and bioavailability in blood plasma. Novel strategies involving CPT pharmacological and low doses combined with nanoparticles have indicated potent anticancer activity in vitro and in vivo. This systematic review aims to provide a comprehensive and critical evaluation of the anticancer ability of nano-CPT in various cancers as a novel and more efficient natural compound for drug development. Studies were identified through systematic searches of PubMed, Scopus, and ScienceDirect. Eligibility checks were performed based on predefined selection criteria. Eighty-two papers were included in this systematic review. There was strong evidence for the association between antitumor activity and CPT treatment. Furthermore, studies indicated that CPT nano-formulations have higher antitumor activity in comparison to free CPT, which results in enhanced efficacy for cancer treatment. The results of our study indicate that CPT nano-formulations are a potent candidate for cancer treatment and may provide further support for the clinical application of natural antitumor agents with passive targeting of tumors in the future. Systematic research in the tumor field has indicated that a set of multifarious processes, including quick spreading and uncontrollable multiplication of abnormal cells, results in the formation of malignant tumors with the potential for metastasis ,2. ThereNatural compounds represent a valued resource in the discovery and development of novel drugs, mainly those used for cancer treatment ,4,5, andCamptotheca acuminata (family: Nyssaceae), a tree native to Tibet and China, which has been widely used in traditional Chinese medicine quinoline), was primarily isolated from Camptotheca acuminata (Nyssaceae) [Ervatamia heyeano [Nothapodytesfeotida [CPT, an indole alkaloid with a pentacyclic ring  . This al heyeano , Nothaposfeotida , and in sfeotida . In 1966sfeotida . CPT hassfeotida .CPT has been indicated to suppress different cancers via various mechanisms both in vitro and in vivo and rapidly entered the clinical examination . The mosDespite the significant antineoplastic activity and an exclusive mechanism of action, CPT displays various unwanted properties which hinder its clinical application. First, the very low water solubility of CPT complicates its administration. Another challenge related to CPT is the α-hydroxy lactone ring (ring E) which opens under physiological conditions, resulting in the CPT carboxylate open form. Carboxylic acid or its sodium salt, even though soluble, has considerably lower antitumor potential as compared to CPT. Furthermore, this ionic form favorably binds to the human serum albumin, lowering the accessible drug concentration ,46.Even though the antitumor properties of CPT stimulated a substantial research interest, other properties of CPT including insecticidal or antiviral activities have also been investigated. Though semisynthetic CPT, such as irinotecan and topotecan, hold their position in chemotherapy, various other anticancer agents are established which are now in different phases of the preclinical or clinical development. Moreover, current research on CPT involves their novel formulations (particularly nano-formulations) for optimizing the stability, delivery, and reducing toxicity .The current study was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines . The purNano-CPT has been indicated to suppress different cancers via various mechanisms, such as growth suppression of malignant cells, induction of apoptosis and cell cycle arrest, and modulation of oxidative stress, angiogenesis, and inflammation. The anticancer effects of CPT nano-formulations in different cancers are provided in the next sections .Bladder cancer is the tenth most frequent cancer in women and fourth in men. The primary treatment for bladder cancer includes intravesical immunotherapy and surgery. Nevertheless, these methods show several undesirable adverse effects ,131. AccPolymeric micelles enhance the drug accumulation in cancer tissues by taking advantage of the increased permeability retention (EPR) impact . In a stGliomas are the most frequent and aggressive brain tumors, and irrespective of progress made in treatment management, they are still limited by several barriers. Therefore, different therapeutic strategies like nano-medicines are needed for treating this disease ,134.One study indicated that treatment of 9L tumor-bearing mice with CPT-loaded amphiphilic β-cyclodextrin NPs inhibited tumor progression and tumor volume, and increased the median survival time. This study indicated that CPT-loaded cyclodextrin NPs arise as potential delivery systems for treating cancer which is indicated to be safe, stable, and effective formulations for CPT delivery .It has been shown that high-dose CPT-loaded NPs (20 mg/kg) inhibited the growth of intracranial GL261 tumors in mice, providing important survival benefits in comparison to low-dose CPT NPs or free CPT . This stAnother study evaluated the anticancer effect of 3 mg/kg CPD@IR780 and CPC@IR780 micelles with or without laser toward Luc-U87 tumor-bearing mice. All the treatments inhibited tumor growth, reduced side effects, and enhanced survival time. However, CPT-S-S-PEG-iRGD@IR780 micelles with laser exhibited superior antitumor activity. Thus, these results indicated that the targeting prodrug system could not only efficiently cross different barriers to reach glioma site but also considerably increase the anticancer impact with laser .Studies showed that treatment of C6 tumor-bearing rats with different CPT-loaded micelles inhibited tumor progression and growth, reduced side effects, and increased median survival rate and therapeutic efficacy ,54. MoreBreast cancer is the utmost frequently diagnosed malignancy and is the second primary cause of mortality globally. Despite these improvements, breast cancer mortality and morbidity is very high . TherefoOne study indicated that in 4T1 tumor-bearing mice, the albumin/CPT-ss-EB nano-complex exhibited efficient tumor accumulation, which subsequently contributed to outstanding therapeutic efficiency . In a stCPT-co-electrospun fibers showed anticancer activity toward 4T1 tumor-bearing mice and led to the suppression of the tumor growth without toxicity [Several studies showed that treatment of 4T1 tumor-bearing mice with different CPT-loaded micelles  inhibited the tumor growth and tumor progression and resulted in enhancement of animal survival rate and therapeutic efficacy ,58,59,60toxicity .Studies indicated that treating 4T1 tumor-bearing mice with various CPT-loaded NPs led to the suppression of tumor growth and reduction in tumor metastases and tumor recurrence ,63,64. MTreating 4T1 tumor-bearing mice with chitosan stabilized CPT nano-emulsions (CHI-CPT-NEs) led to the reduction of tumor growth and volume and enhancement of therapeutic efficacy . AdditioIn another study, the in vivo synergistic antitumor efficiency of HSD NGs was assessed in 4T1 tumor-bearing mice, and results indicated that HSD NGs enhanced apoptosis and tumor accumulation, and suppressed tumor growth and volume .Treating MCF-7 tumor-bearing mice with various CPT-loaded micelles led to the strong inhibition of the tumor growth without toxicity ,70,71. Mg-4-HC increased the synergistic antitumor efficacy and apoptosis in rats bearing MCF-7 tumor cells [Several studies showed that treatment with different CPT-loaded nano-formulations led to tumor elimination and inhibited tumor growth and volume in MCF-7 tumor-bearing mice ,74,75,76or cells . Anotheror cells .Studies showed that an increase in survival rate and tumor inhibition has been observed in MDA-MB-231 tumor-bearing mice after treatment with CPT-HGC NPs  and CPT-CPTNV-Pmic) exhibited considerable anticancer activities against the Ehrlich ascites carcinoma (EAC) mice model and reduced tumor necrosis and tumor growth [3 PA nanofibers was assessed in BT-474 tumor-bearing athymic nude mice, and results demonstrated that these nanofibers inhibited tumor growth and tumor progression [Treatment of EMT6 tumor-bearing mice with 1 mg/kg CPT/DOX-CCM and CPT/DOX-NCM, for 24 days led to tumor inhibition and reduced tumor size and volume . CPT-loar growth . In anotgression . Such naCervical cancer is the second highest cause of mortality amongst women . Even thx-PEG-FA NPs andPDA@PCPT NPs) led to the suppression of tumor progression and tumor growth and increased the therapeutic efficiency in HeLa tumor-bearing mice [Studies showed that treatment with different CPT-loaded NPs  is one of the utmost common malignant tumors. The main approaches for the treatment of CRC are surgery, chemotherapy, and radiotherapy . Yet, duOne study showed that transformative CPT-ss-EB nanomedicine considerably suppressed tumor progression and drastically decreased side effects in HCT116 tumor-bearing mice . AnotherIn one study the antitumor activity of CPT&Ce6 and HBPTK-Ce6@CPT with or without laser irradiation in HT29 tumor-bearing nude mice has been evaluated. All the treatments inhibited tumor growth and volume. However, HBPTK-Ce6@CPT with 660 nm laser irradiation had greater anticancer activity in comparison to other groups .Studies showed that treatment of HCT116 tumor-bearing mice with different CPT-loaded NPs resulted in the elimination of tumor, reduction of tumor growth and volume, tumor recurrence, and increase of therapeutic efficacy and apoptosis ,95,96,97Treatment of HT-29.Fluc tumor-bearing mice with 0.8 mg/kg SNP-CPT-Cy5.5 and SNP-CPT NPs led to the tumor elimination and inhibited tumor growth and progression, while considerably decreasing the systemic toxicity associated with CPT administration. These results demonstrate that the SNP-CPT NPs can be used as a potent drug delivery system for CPT-based anticancer treatments .In a study by Yao et al. , the in Gd(DTPA-CPT) NPs showed significant cytotoxicity against LoVo tumor-bearing nude mice and led to the tumor growth suppression with reduced adverse effects and insignificant chronic toxicity . AnotherSeveral studies indicated that treating C26-tumor-bearing mice with different CPT nano-formulations resulted in the elimination of tumor growth and volume, enhancement of therapeutic efficacy, and reduction in tumor progression ,105,106.Hepatocellular carcinoma (HCC) is the second leading cause of death in the world. Irrespective of the advancement in existing HCC treatments, there has been a continuous increment in the incidence rate of this cancer .In a study by Wen et al. , the in Moreover, studies showed that treatment with different CPT prodrugs (CCLM and RGD-prodrug) led to tumor elimination and suppressing tumor progression and growth in HepG2 and H22 tumor-bearing mice ,110. TheIn one study the antitumor activity of IR780-LA/CPT-ss-CPT NPs were investigated towards Hep1–6 tumor-bearing mice. It was noticed that the tumor volume and growth in all groups were suppressed. Nevertheless, the tumor growth in the IR780-LA/CPT-ss-CPT NPs + laser group was the most strictly suppressed. Consequently, the IR780-LA/CPT-ss-CPT NPs were indicated to be a brilliant fluorescence imaging-guided, redox-responsive, and increased synergistic chemo-photothermal therapy nanoplatform toward tumors .Treatment of HepG2 tumor-bearing mice with P (CPT-MAA) nanogels without SeS and P(CPT-MAA) prodrug nanogels exhibited superior antitumor activity without observed side effects. However, P(CPT-MAA) prodrug nanogels indicated the greatest efficacy in tumor growth suppression. Henceforth, the P(CPT-MAA) nanogels might be a potent delivery system for anticancer agents .JP@EF and JNM@EF showed cytotoxic effects against H22 tumor-bearing mice, while the most considerable suppression of tumor growth was identified for JNM@EF treatment. It was demonstrated that the JNMs self-propelled tissue distribution and gradual release increased the tumor growth suppression .In another study, the anticancer effects of UCNP@mSiO2–NBCCPT @(DHMA)/β-CD-PEG, UCNP@mSiO2-NBCCPT@(DHMA)/β-CD-PEG-LA, and UCNP@mSiO2-NBCCPT/β-CD-PEG were assessed in HepG2 tumor-bearing mice. All the treatments inhibited tumor growth, while UCNP@mSiO2-NBCCPT/β-CD-PEG-LA@DHMA had greater antitumor activity and ameliorated side effects. Therefore, it showed more beneficial for prolonging the survival of tumor-bearing mice . OverallLung cancer is one of the leading causes of tumor-related mortality in the world . The higEven though CPT is a renowned antitumor agent, it typically needs high and multiple doses for achieving a satisfactory therapeutic impact. One study investigated the antitumor impact of CPT-loaded PPBS NPs in LLC tumor-bearing mice. Results of this study indicated that CPT-loaded PPBS NPs reduced the tumor volume and had a longer circulation time, and substantially improved anticancer efficiency in vivo compared to free CPT .NCssG NPs and CPT-ss-GEM nanowires (CssG NWs) showed cytotoxic effects against A549 tumor-bearing mice and suppressed tumor growth and volume. Results also showed that with the same amount of CPT-ss-GEM prodrug, NCssG NPs showed much higher in vivo tumor inhibition efficiency than CssG NWs. Due to the superiority of this combo-nanomedicine, such as EPR effect, synchronous dual drug action, prolonged blood circulation, very potent antitumor efficiency was achieved both in vitro and in vivo .Studies showed that treatment of A549 tumor-bearing nude mice with different nano-formulations of CPT resulted in tumor elimination, reduction in tumor growth and volume, and enhancement of therapeutic efficacy ,118. TheAnother study indicated that treatment with ZTC-NMs delayed tumor growth and prolonged the survival time in A549 tumor-bearing mice. Also these results showed that ZTC-NMs had a greater antitumor activity, which can be ascribed to the tumor-targeted drug delivery .In one study the antitumor effect of Ce6-CPT-UCNPs with or without laser irradiation was evaluated in NCI-H460 tumor-bearing nude mice. Results showed that treatment with Ce6-CPT-UCNPs + laser irradiation inhibited tumor growth, tumor recurrence, and metastasis. In contrast, treatment with Ce6-CPT-UCNPs and no laser irradiation did not show antitumor activity, because CPT and Ce6 in the Ce6-CPT-UCNPs cannot be released out, and cannot eliminate cancer cells .Treatment of NCI-H460 tumor-bearing nude mice with a new mitochondria-targeting drug delivery system, ZnPc/CPT-TPPNPs or ZnPc/CPT-NH2NPs led to the suppression of tumor growth, tumor recurrence, and metastasis and did not result in considerable side effects in vivo. These results demonstrated that surface modification of the NPs with triphenylphosphine cations simplified effective subcellular delivery of the photosensitizer to mitochondria .In another study, the in vivo anticancer efficiency of 2OA-CPT/NAs and OA-CPT/NAs was compared using the LLC tumor-bearing mice. It is found that treatments with different CPT formulations led to a considerably delayed tumor progression. However, in comparison with OA-CPT/NAs, 2OA-CPT/NA exhibited a greater potent anticancer activity. These results revealed the critical role of DHP hydrophobicity in impacting the DHP NA in vivo anticancer efficacy .Ovarian cancer represents a group of neoplasms, and it is one of the utmost lethal female reproductive system tumors . The conIn one study CPT was encapsulated into plain NOBs (NOB–CPT) and ZH2-displayed NOBs (ZH–NOB–CPT) and then administered to SKOV3 tumor-bearing nude mice. Results showed that treatment with 0.5 mg/kg ZH–NOB–CPT significantly inhibited tumor growth and volume. In contrast, NOB–CPT-treated mice did not show any antitumor effects. This study indicates that ZH2-tagged NOBs selectively deliver CPT into the human epidermal growth factor receptor 2/neu-positive cancerous site. Taken together, the result shows the NOB’s potential for targeted delivery of hydrophobic drugs .Pancreatic cancer is a disease with a high mortality rate and a very poor prognosis. Various biological and physical barriers make this tumor very hard to treat with conventional chemotherapy. Hence, more effective strategies, such as nano-therapies, are crucial to fulfill the immediate necessity for more effective pancreatic cancer treatment ,147.One study evaluated the therapeutic effects of EGGPTCPT, PEGPTCPT, and GSHPTCPT in BxPC-3 tumor-bearing mice. All the treatments resulted in tumor inhibition, while GSHPTCPT displayed considerably greater anticancer activity than PEGPTCPT and EGGPTCPT. There was no considerable difference between PEGPTCPT and EGGPTCPT in terms of tumor weight and volume. This study indicates the high efficacy of an active tumor penetrating dendrimer-drug conjugate for PDA therapy .Another study showed that treatment with 2 mg/kg CPT-loaded αDR5-NPs and CPT-loaded nude NPs noticeably suppressed tumor growth rates and induced tumor regressions in MIA PaCa-2 and PANC-1 xenografts in mice. The effects were more considerable in the MIA PaCa-2 cells where delivery of CPT-loaded αDR5-NPs resulted in considerable tumor regressions, whereas the CPT-loaded nude NPs only resulted in growth retardation. These results indicate the CPT-loaded αDR5-NPs potential for pancreatic cancer treatment .In a study by Wang et al. , the DPPProstate cancer is one of the most common cancers in men . The comOne study indicated that treatment of U14 tumor-bearing nude mice with CPT-HA and CPT-HA@IR825 with or without laser led to tumor elimination. In vivo results demonstrate that after treating tumors with CPT-HA or CPT-HA@IR825 without laser, the tumor volume was only partially inhibited. In sharp contrast, the tumor growth from the CPT-HA@IR825-treated mice upon laser irradiation has been effectively suppressed, indicating that the multifunctional polymeric prodrug NPs are very effective for cancer treatment, while having reduced cytotoxicity to the healthy organs .Another study indicated that treatment with P(OEGMA-co-CPT-co-G3-C12) or P(OEGMA-co-CPT) for 32 days inhibited tumor volume and tumor growth in DU145 tumor-bearing BALB/c mice. Results of this study indicated that P(OEGMA-co-CPT-co-G3-C12) had a greater antitumor activity compared to P(OEGMA-co-CPT). Furthermore, P(OEGMA-co-CPT-co-G3-C12) indicated minimal toxicity and improved antitumor activity compared to free CPT. Henceforth, P(OEGMA-co-CPT-co-G3-C12) can be a potent drug in androgen-independent prostate cancer treatment .Melanoma is the primary cause of death from skin cancer . GeneralIn a study by Hu et al. , nude miThe plasma drug time-concentration profile of CPT was constructed after the oral administration of 5 mg/kg pure CPT suspension and CPT encapsulated poly (methacylic acid-co-methyl methacrylate) nano-formulation to rats. The results demonstrated a considerable difference between the CPT nano-formulation and free CPT pharmacokinetic profiles. After oral administration of free CPT, the drug was detected rapidly in plasma in the initial hours, ascribed to the greater CPT permeability coefficient in the upper GIT. Subsequently, the drug-plasma concentration reduced rapidly to undetectable levels after 8 h. In the case of CPT nano-formulation, the maximum CPT level was reached at 12 h after oral administration as the polymer present in the nano-formulation enhanced the CPT residence time and then gradually reduced over the next 12 h, which demonstrated the released drug prolonged residence time in the colon with drug slow leaching to systemic circulation because of low permeability and compromised surface area ,153.After a single oral administration (1.5 mg/kg) of CPT and CPT nanocrystals in Sprague-Dawley rats, it was reported that in comparison to CPT salt solution, CPT nanocrystal’s area under the curve (AUC) value was lower, but the distribution half-life (t1/2α) was higher, showing prolonged circulation via the nanocrystals. It seems to be plausible that due to the poor solubility, CPT nanocrystals gradually dissolved and released free drug molecules . TherefoFor evaluating the subacute toxicity of CPT NPs, ICR mice were administered with 0.4 mL/20 g. body weight NPs through i.p. injection. There was no treatment-related death in the CPT NPs treated mice at any of the doses tested. Nevertheless, the free CPT group body weight was considerably lower than information from the control group and the two other CPT groups .In an inorganic–organic model, CPT was primarily incorporated into micelles derived from negatively charged biocompatible surfactants, such as sodium cholate, and these negatively charged micelles were next encapsulated in NPs of magnesium–aluminum layered double hydroxides (LDHs) via an ion exchange process. Although these nano-complexes exhibited lower cytotoxicity than CPT alone, they act as a potent biocompatible model for delivering CPT, enabling the administration of drugs in a dose-controlled manner .After CPT-loaded GNR@SiO2−tLyP-1 treatment, the systemic toxicity of CPT on human mesenchymal stem cells was reduced, since the GNR@SiO2−tLyP-1 selectively penetrated the cancer cells, and CPT was released into the blood or culture medium .Natural products with remarkable chemical diversity have been examined in the treatment of human malignancies and particularly in cancer treatment. CPT, a natural plant alkaloid, has indicated strong antitumor activities via targeting intracellular Topo I. CPT alone or in combination with other anticancer drugs might be beneficial for cancer treatment. Studies indicated that CPT nano-formulations have higher antitumor activities in comparison to free CPT and lead to better bioefficacy for the treatment of cancer. CPT can affect different cancer types, including bladder, brain, breast, cervical, colon, leukemia, liver, lung, melanoma, and prostate cancer. It has been shown that CPT employs multiple mechanisms for suppressing cancer initiation, progression, and promotion through modulating different dysregulated signaling cascades implicated in proliferation, invasion, inflammation, cell survival, metastasis, and apoptosis .The promise that CPT holds as an antitumor agent in therapy is restricted by factors that include lactone ring instability and water insolubility, which restricts the drug oral solubility and bioavailability in blood plasma. Novel approaches including pharmacological and low doses of CPT in combination with NPs have indicated potent anticancer potential in vivo. The results of our study indicate that CPT nano-formulations is a potential candidate for cancer treatment, and might provide further support for developing nano-CPT as a promising agent for cancer treatment.


Self-collected specimens can expand access to SARS-CoV-2 testing. At a large inner-city hospital 1,082 participants self-collected saliva and anterior nasal swab (ANS) samples before healthcare workers collected nasopharyngeal swab (NPS) samples on the same day. To characterize patient preferences for self-collection, this investigation explored ability, comfort, and ease of ANS and saliva self-collection for SARS-CoV-2 testing along with associated patient characteristics, including medical history and symptoms of COVID-19. With nearly all participants successfully submitting a specimen, favorable ratings from most participants (at least >79% in ease and comfort), and equivocal preference between saliva and ANS, self-collection is a viable SARS-CoV-2 testing option. As the COVID-19 pandemic continues into a second year , travel This analysis used data collected as part of a cross-sectional investigation comparing performance of self-collected saliva and ANS to healthcare worker-collected NPS on the same day from patients at Grady Memorial Hospital in Atlanta, GA, as previously described . BrieflyParticipants self-collected saliva and ANS using standardized infographics for instructions . Saliva Among 1082 participants, 47.8% were female, mean age was 50 years (IQR 38–61), 81.1% were non-Hispanic Black, 80.9% reported an underlying medical condition associated with severe COVID-19 outcomes , and 18.Overall, 48.5% of participants preferred saliva collection (over ANS), 42.7% preferred ANS , and 4.5% had no preference between the two self-collected samples . Saliva Nearly all participants were able to provide a sufficient saliva (95.5%) and ANS (99.4%) specimen for testing . SixteenThis investigation found that self-collected specimens for SARS-CoV-2 testing were an acceptable option to participants; nearly all were able to provide self-collected saliva and ANS specimens and rated the experience favorably in at least one category. High collection success rates indicate that no person should be automatically excluded from public health self-collection programs based solely on demographic factors. Those with potential dexterity impairment, including neurological disorders or arthritis, were just as likely to have success with self-collection. Importantly for reaching those disproportionally affected by the COVID-19 pandemic, this investigation was conducted in a public hospital and included many participants at increased risk for severe COVID-19 disease –18.Since instructions for self-collection were given in written infographic format and did not rely on interviewers or healthcare personnel for teaching, self-collection testing programs need not be limited to healthcare settings. Self-collection testing could therefore potentially preserve valuable healthcare resources, including facilities, personnel, and PPE. Additionally, higher favorable ratings for both self-collected specimen types in asymptomatic individuals and those in pre-procedure screening suggest that self-collected specimens might be even more favorable in screening programs compared to clinical, diagnostic testing situations. Notably, although our study used SARS-CoV-2 RT-PCR testing, ANS are included as approved specimens for several SARS-CoV-2 antigen tests with Emergency Use Authorizations approved by the US Food and Drug Administration. The ability to screen with self-collected rapid antigen tests is an important tool for expanding access to testing, especially for situations where frequent testing has been proposed, such as in school settings , 20. SelThe very small subset of participants with difficulty providing saliva were older and possibly in poorer health; they were however able to provide ANS specimens, so offering both modalities in a testing program for this population, such as in a nursing home, might increase successful participation. Saliva was the preferred method of self-collection by a small margin; those with neurological disorders were the only group with a statistically significant preference for ANS. When attempting to broaden access with a self-collected specimen diagnostic program, either self-collection modality would likely be similarly acceptable in many settings, given the almost equal preference between the two.This investigation has several limitations. First, despite written infographics being the main source of instruction, interviewers were present and an element of bedside manner or outside coaching may have contributed to ratings and preferences; therefore the experience may not translate from this element of interviewer presence to true unsupervised self-collection. Second, language translation services and Spanish instructional handouts were used, but communication difficulties  were not assessed; these may have affected understanding of the instructional handout despite being mostly pictorial. Third, 50 milliliter conical centrifuge tubes were used for saliva collection. These have a narrow mouth, which may have affected saliva-collection ratings. Fourth, the diagnostic performance report of this same investigation demonstrated that the self-collected saliva and ANS specimens had overall lower sensitivity than NPS specimens , and 59% (95%CI: 47–70%), respectively) , which mAccessible SARS-CoV-2 diagnostic testing is important for routine patient care and resumption of in-person activities, and self-collected specimens can help expand access while reducing personnel and resource burdens on the healthcare system.S1 File(PDF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


The selected studies reported that micro-CT could successfully detect several lung diseases providing three-dimensional images of greater detail and resolution than routine optical slide microscopy, and could additionally provide valuable volumetric insight in both restrictive and obstructive lung diseases. In conclusion, micro-CT-based volumetric measurements and qualitative evaluations of pulmonary tissue structures can be utilized for the clinical management of a variety of lung diseases. With micro-CT devices becoming more accessible, the technology has the potential to establish itself as a core diagnostic imaging modality in pathology and to enable integrated histopathologic and radiologic assessment of lung cancer and other lung diseases.Micro-computed tomography (micro-CT) is a promising novel medical imaging modality that allows for non-destructive volumetric imaging of surgical tissue specimens at high spatial resolution. The aim of this study is to provide a comprehensive assessment of the clinical applications of micro-CT for the tissue-based diagnosis of lung diseases. This scoping review was conducted in accordance with the PRISMA Extension for Scoping Reviews, aiming to include every clinical study reporting on micro-CT imaging of human lung tissues. A literature search yielded 570 candidate articles, out of which 37 were finally included in the review. Of the selected studies, 9 studies explored via micro-CT imaging the morphology and anatomy of normal human lung tissue; 21 studies investigated microanatomic pulmonary alterations due to obstructive or restrictive lung diseases, such as chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and cystic fibrosis; and 7 studies examined the utility of micro-CT imaging in assessing lung cancer lesions ( Micro-computed tomography (micro-CT) constitutes a novel non-destructive ex vivo medical imaging modality that enables volumetric imaging of intact surgical lung tissue specimens at high spatial resolution and is thereby capable of providing microscopic insight into alveolar anatomy and pathology . Due to Similar to CT imaging in radiology, micro-CT imaging relies on an X-ray source and detector assembly in combination with computational image reconstruction algorithms to acquire a series of 2D planar, cross-sectional digital images of the imaging target, which can be computationally processed to generate 3D volumetric models . In contHowever, evidence about the role and utility of micro-CT imaging in the diagnosis and management of lung disease is scarce. To evaluate potential clinical applications of micro-CT imaging of surgical lung tissue specimens, we systematically reviewed the scientific literature and collected all publicly available evidence. Based on our findings, we foresee a pivotal future role of this emerging imaging modality in the interdisciplinary diagnosis of lung disease.ClinicalTrials.gov, and Cochrane CENTRAL until 12 February 2021. Our search included the medical subject heading terms (MeSH): (“micro-CT” OR “X-ray micro-tomography” OR “CT micro-tomography”) AND (“lung*” OR “pulmonary”). Additionally, the PROSPERO database (www.crd.york.ac.uk/prospero) was investigated on 12 February 2021 to identify relevant systematic reviews in order to ensure that there is no other ongoing systematic review on this subject.A scoping review methodology was chosen to achieve this article’s goal to provide insight into the clinical applications of micro-CT imaging for the diagnosis of pulmonary diseases. This was conducted according to the PRISMA Extension for Scoping Reviews (PRISMA-ScR) . SpecifiArticles selected by this search were included in this review if (1) they reported on the utilization of micro-CT in pulmonary imaging and if (2) they were published in peer-reviewed scientific journals. An article was excluded if it met at least one of the following criteria: (1) preclinical/animal research, (2) not available in English, and (3) not original research, but review or editorial article type.After importing all articles into the Rayyan reference management program,  three reFor each eligible article, data were extracted based on (i) country of the author group and year of publication; (ii) study design and population; (iii) objective and main outcome of the study; (iv) type of pulmonary disease investigated; and (v) micro-CT imaging details . The tabular and descriptive presentation of our results was based on the type of pulmonary tissue or disease assessed: (i) physiological lung tissue; (ii) chronic obstructive pulmonary disease (COPD); (iii) idiopathic pulmonary fibrosis (IPF); and (iv) others: cancer, congenital anomalies, infections, or lung transplantation-related diseases. Studies reporting micro-CT scanning of lung specimens in more than 1 pulmonary field were inserted in both categories.An assessment of the quality of the included studies was conducted, based on the Newcastle-Ottawa Quality scale . The queOur initial literature research in the aforementioned databases resulted in 570 potentially eligible articles. The number of studies that remained after each filtering step is depicted in The quality of the included studies was systematically evaluated in accordance with the Newcastle–Ottawa quality scale, as illustrated in n = 17) used micro-CT imaging to assess the pathological changes in lung specimens of patients with either COPD , Nikon HMX Metrology (n = 8), Explore Locus (n = 3), IspeXio SMX (n = 3), Med X-Alpha (n = 2), and a variety of other models (n = 7).Nineteen studies were performed in Europe , 13 in North America (USA and Canada), and 5 in Asia . The number of the samples tested ranged from 2 to 530 lung specimens . Cryo-miThis scoping review presents a comprehensive overview of the current landscape of clinical applications of pulmonary micro-CT imaging . Micro-CIn some application areas, micro-CT has enabled novel insights into the 3D structural alterations in pathological lung specimens. In COPD, for example, micro-CT imaging has made it possible to quantify the loss of terminal bronchioles in early-stage COPD, which has been proposed to pave the way for the emphysematous destruction of the alveoli ,37. ThesFurthermore, micro-CT imaging can be used along with micro Optical Coherence Tomography (micro-OCT), CT imaging, and slide microscopy to comprehensively assess pathological changes in IPF ,55,56. MMicro-CT allows non-destructive 3D visualization and assessment of lung specimens routinely prepared in pathology, including fresh-frozen tissues as well as formalin-fixed, paraffin-embedded tissue blocks ,44. The Micro-CT has also been employed successfully for the study and diagnosis of lung cancer. In particular, the technology facilitates the identification of lymphatic heterogeneity between normal and malignant lung tissues, giving insight into the lymphatic metastatic spread in lung cancer . AdditioApart from the ability to evaluate surgical margins and lesion size, high-resolution whole-lesion morphologic acquisition could also supplement slide-based histopathology diagnosis, for example by directing the pathologist toward histology sections that are most likely to influence intraoperative decision-making. It should also be noted that conventional histology sectioning could miss significant histopathologic features due to sparse sampling of the specimen. Micro-CT could also expedite the analysis, especially when performed after resection of specimens with metallic fiducial markers placed preoperatively. Optimization of the parameters affecting the perioperative use of micro-CT as well as larger scale studies could lead to the integration of micro-CT into the clinical workflow of lung cancer resection, which could significantly bolster intraoperative decision-making.Interestingly, micro-CT has been also proposed as a tool for the investigation of transplantation-related disease characteristics and for the selection of candidate lung transplants. Firstly, micro-CT can effectively quantify terminal bronchioles and complement histopathology in the identification of pathological alterations in BOS and RAS . Also, iThe emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing COVID-19 has resulted in hundreds of thousands of casualties worldwide, thereby prioritizing the COVID-19-related research for the rationalization of new treatment strategies. Micro-CT could emerge as another tool in researchers’ arsenal in studying the pathogenesis of the disease and assessing disease severity, as demonstrated in two recent preclinical animal-based studies that investigated the inflammatory response and the extent of lung injury induced by SARS-CoV-2 infection ,59. UtilOut of the aforementioned applications of micro-CT scanning, its potential involvement in perioperative lung cancer management could be more extensively described. Specifically, besides the perioperative evaluation of tumour size, location, and resection margins within resection specimens, micro-CT could also facilitate identification and quantification of tumour spread through air spaces (STAS), all without compromising specimen integrity. The concept of STAS has first been described in 2008 as a negative prognostic indicator in lung adenocarcinoma  and consMicro-CT is a promising novel non-destructive imaging technique, which allows the acquisition of volumetric images of lung tissue specimens at high throughput and high spatial resolution without requiring additional specimen preparation. The technology has already been employed in several clinical studies to visualize, measure, and evaluate the characteristics of both normal and diseased lungs at microscopic resolution in 3D. Especially in the context of chronic obstructive and restrictive lung disease, lung cancer, and lung allograft rejection, micro-CT imaging has provided unprecedented insight into the underlying pathological microanatomic changes and has the potential to improve the diagnosis and therapy of patients suffering from these diseases. Given its favourable imaging properties, we anticipate that micro-CT will gain widespread clinical adoption, fill the resolution gap between in vivo CT imaging of the entire lung in radiology and in vitro slide microscopy imaging of lung tissue section specimens in pathology, and ultimately enable 3D pathology across multiple length scales and across disciplinary boundaries.


These variants were found to be associated with pathways governing host immunity, such as interferon, interleukin and cytokine signalling, and known COVID-19 comorbidities, such as obesity and cholesterol metabolism. Further, our ancestry analysis revealed that the asymptomatic COVID-19 patients possess discernibly higher proportions of the Ancestral North Eurasian (ANE) and Eastern Hunter-Gatherer (EHG) ancestry, which was introduced to Europe through Bell Beaker culture (Yamnaya related) and lower fractions of Western Hunter-Gatherer (WHG) ancestry, while severely symptomatic patients have higher fractions of WHG and lower ANE/EHG ancestral components, thereby delineating the likely ancestral differences between the two groups.The coronavirus disease (COVID-19) caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is characterized by a wide spectrum of clinical phenotypes ranging from asymptomatic to symptomatic with mild or moderate presentation and severe disease. COVID-19 susceptibility, severity and recovery have demonstrated high variability worldwide. Variances in the host genetic architecture may underlie the inter-individual and population-scale differences in COVID-19 presentation. We performed a genome-wide association analysis employing the genotyping data from AncestryDNA for COVID-19 patients of European descent and used asymptomatic subjects as the control group. We identified 621 genetic variants that were significantly distinct between asymptomatic and acutely symptomatic COVID-19 patients (multiple-testing corrected  It is caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)  = 0.79 (95% CI: 0.62–1.02), |Z| = 1.79, p-value = 0.07). While this might be attributed to the smaller sample size of asymptomatic versus severely symptomatic patients, it could still be biologically meaningful. We did not find any association between BMI and the severity of COVID-19 (Odd’s Ratio [BMI < 25] = 0.91 (95% CI: 0.69–1.2), |Z| = 0.66, p-value = 0.51). We attribute the lack of association between BMI and the severity of COVID-19 to the age and gender variation in the dataset, both of which have previously been linked to BMI . R. RqpAdm e former  and hencThe findings in the current study may have been influenced by the limited availability of genetic data for COVID-19 patients. Among the SARS-CoV-2 infected cohort, we sub-categorized patients as asymptomatic, mild, moderate or severe and the rest as unknown based on a self-reported questionnaire. Since the aforesaid categories were delineated solely based on the questionnaire, we cannot exclude the likelihood of miscategorization, which might have influenced our results. Further, the unavailability of the genomic data for COVID19 patients in the ICU or those who may have succumbed might cause some discrepancies in the final outcomes of our study. Finally, we note that, as in the case of previous association studies, our GWAS findings may not be exclusively ascribed to SARS-CoV-2 infections alone. However, we surmise that utilizing asymptomatic COVID-19 patients as controls improves the probability of the genetic variants identified herein to being associated with the degree of COVID-19 manifestation specifically, as these are significantly different between the severely symptomatic and asymptomatic COVID-19 subjects.Overall, our findings elucidate the striking genetic differences between asymptomatic and severely affected patients infected with SARS-CoV-2 infection. Expanding this approach to include whole-genome sequencing data and increasing the power of analysis by employing a large number of individuals in various categories of SARS-CoV-2 infection severity accentuates the potency for uncovering novel genetic variants that may be associated with severe COVID-19 in future, thereby likely identifying cellular pathways that may be targeted to develop or improve therapeutics. Further, using a population genetics driven approach such as ours in diverse ancestries will provide the opportunity to interrogate population and ancestry specific genetic factors that may govern susceptibility to severe COVID-19 and may uncover clinically actionable and more efficacious population-specific drug targets.The current study shines a light on the striking differences in the genetic architecture between asymptomatic and severely affected COVID-19 patients of European descent. While asymptomatic individuals contain significantly larger ANE/EHG ancestral fractions, patients with severe clinical manifestations possess dominant WHG fractions. Host pathways governing immunity such as innate and adaptive immune system, IFN, IL, cytokine pathways, antigen processing and SNVs related to comorbidity attributes, such as obesity-related traits, cardiovascular disorders, lung function and smoking, were discerned to vary significantly between severe and asymptomatic COVID-19 patients.


Clavispora NRRL Y-50464, is one solution. NRRL Y-50464 is fast-growing and robust, and tolerates inhibitory compounds 2-furaldehyde  and 5-hydroxymethyl-2-furaldehyde (HMF) associated with lignocellulose-to-fuel conversion. It produces three forms of β-glucosidase isozymes, BGL1, BGL2, and BGL3, and ferment cellobiose as the sole carbon source. These β-glucosidases exhibited desirable enzyme kinetic parameters and high levels of enzyme-specific activity toward cellobiose and many oligosaccharide substrates. They tolerate the product inhibition of glucose and ethanol, and are stable to temperature and pH conditions. These characteristics are desirable for more efficient cellulosic ethanol production by simultaneous saccharification and fermentation. NRRL Y-50464 provided the highest cellulosic ethanol titers and conversion rates at lower cellulase loadings, using either pure cellulose or agricultural residues, as so far reported in the literature. This review summarizes NRRL Y-50464 performance on cellulosic ethanol production from refined cellulose, rice straw, and corn stover processed in various ways, in the presence or absence of furfural and HMF. This dual functional yeast has potential to serve as a prototype for the development of next-generation biocatalysts. Perspectives on continued strain development and process engineering improvements for more efficient cellulosic ethanol production from lignocellulosic materials are also discussed.Reducing the cost of cellulosic ethanol production, especially for cellulose hydrolytic enzymes, is vital to growing a sustainable and efficient cellulosic ethanol industry and bio-based economy. Using an ethanologenic yeast able to produce hydrolytic enzymes, such as  In a conventional simultaneous saccharification and fermentation (SSF) process for cellulosic ethanol production, cellulase hydrolyzes cellulose into oligoglucans and cellobiose, and additional β-glucosidase converts cellobiose into glucose for yeast fermentation. Beta-glucosidase  hydrolyzes nonreducing β-D-glucosyl residues from glycosides and β-linked oligosaccharides, releasing glucose. Enzyme costs for cellulosic ethanol are approximately 10 times greater than conventional starch-based fermentations using amylases , 41β-gluβ-glucosidase , 41β-gluIn addition to tolerance to glucose inhibition, BGL2 is highly resistant to product inhibition caused by ethanol . Its speHydrolysis of cellulose releases a mixture of diversified oligosaccharides. In addition to assay using the chromogenic substrate pNPG, specific activity of BGL2 and BGL3 was evaluated toward 14 purified oligosaccharides . These sβ-glucosidase and ferment cellobiose, as well as glucose, to ethanol. The yeast is also able to achieve higher ethanol titers from refined cellulose with a decent productivity. Its ethanol production properties on agricultural materials, including industrial by-product corncob residue, rice straw, DOE-NREL-pretreated corn stover, and conventional corn stover, are exceptional compared to other reported yeast strains in terms of efficiency and economics. Most significantly, the ethanol conversion rate of NRRL Y-50464 is outstanding among other available β-glucosidase producing strains. Identification of a β-glucosidase family containing at least three members of BGL1, BGL2, and BGL3 with desirable enzymatic characteristics further supports its dual function of enzyme hydrolytic functions in addition to ethanol production capability. While fast-growing and robustness of a microbial strain are desirable characteristics for industrial applications, care should be taken to prevent potential contaminations of unintended applications since NRRL Y-50464 grows faster than most yeast strains including S. cerevisiae. It needs to point out that the current ethanol titer by NRRL Y-50464 is still low and not ready for industrial applications. A significant strain improvement is needed to further increase its ethanol conversion capability.Beta-glucosidase as an important component of cellulase is required for end-hydrolysis of cellulose-to-ethanol production by SSF. Reducing enzyme costs remains a significant challenge for commercial production of advanced biofuels. Microbes that natively produce hydrolytic enzymes typically are not homo-ethanol fermenters. The fast-growing and inhibitor robust yeast strain NRRL Y-50464 is rare in its ability to produce S. cerevisiae. Unlike well-characterized S. cerevisiae, the genomic background of Clavispora NRRL Y-50464 is less known. Since the detoxification mechanisms of NRRL Y-50464 are similar to those observed in S. cerevisiae, it may not be a far-reachable goal to enable a xylose-to-ethanol pathway in NRRL Y-50464 with appropriate modifications.NRRL Y-50464 can utilize xylose but produces xylitol rather than ethanol. This is a major defect of this strain for cellulosic ethanol production since lignocellulosic biomass contains a large portion of xylose. It is an important area, which needs to be improved in order to take a full advantage of this strain for cellulosic ethanol production using hydrolysates. Genetic engineering to redirect xylose-to-ethanol conversion pathway would significantly enhance its utilization of mixed C-5 and C-6 sugars, such as accomplished in S. cerevisiae to enable its xylose utilization [YXI, a synthesized yeast codon-optimized xylose isomerase gene, in S. cerevisiae achieved a constitutive expression for the industrial yeast. Use YXI, alone with a set of exogenous xylose transporter genes and downstream facilitating genes; several new genotypes were obtained to significantly improved xylose uptake and utilization for cellulosic ethanol production [S. cerevisiae [YXI initiated xylose metabolic pathway and subsequently facilitated interactions with the critical nonoxidative pentose phosphate pathway branch for enhanced xylose uptake and utilization [There are numerous engineered examples of lization –55. Chrooduction , 53, 56.β-glucosidase and related genes, not only benefits the improvement of NRRL Y-50464, but also can be applied to other yeast for strain enhancement and development. With rapid advances of science and biotechnology such as genome editing and CRISPR tools, it is expected that more desirable strains can be accomplished in the future. NRRL Y-50464 can serve a valuable new genetic resource in this regard.Currently, the genome sequence of NRRL Y-50464 is not available yet and limited genetic background is known. Genomic study on this strain will reveal fundamental mechanisms of its dual functions aiding its extended improvement and applications. Characterization of the genetic background of NRRL Y-50464, especially for those Further process engineering is also needed to improve the conversion efficiency of the bioreactor. Traditional bioreactor is designed for liquid fermentation with bottom stirring, which is not ideal for ethanol production from high solids of pretreated lignocellulosic feedstock. Bottom stirring limits conversion efficiency significantly when solids loading levels are greater than 15% . High soLignin has been well known as a major factor limiting enzymatic biomass deconstruction and higher titers of cellulosic ethanol production, and it was also observed from corn stover conversion using NRRL Y-50464 , 57. MilThus, an integrated improvement is needed, which consists of strain performance, enzyme hydrolytic efficiency, more efficient pretreatment methods, and proper process engineering, to achieve a low-cost cellulosic ethanol production from lignocellulosic materials., 58, 59.