The inducible cytoprotective enzyme heme oxygenase-1 (HO-1) has gained significant recognition in recent years for mediating strong cellular resistance to a broad range of viral infections, regardless of the type of viruses, viral strains, or mutants. HO-1 is not a typical antiviral agent that targets any particular pathogen. It is a “viral tropism independent” endogenous host defense factor that upon induction provides general cellular protection against pathogens. By virtue of HO-1 being widely distributed intracellular enzyme in virtually every cell, this unique host factor presents a novel class of generic host defense system against a variety of viral infections. This Viewpoint proposes pharmacological evaluation of the HO-1-dependent cellular resistance for its potential in mitigating infections by deadly viruses, including the current severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), its variants, and mutants. HO-1-dependent cellular resistance against SARS-CoV-2 can complement current medical modalities for much effective control of the COVID-19 pandemic, especially with constantly emerging new viral variants and limited therapeutic options to treat SARS-CoV-2 infection and associated severe health consequences. Coronaviruses constitute a large group of viruses known to cause illnesses similar to common cold and more severe diseases, including severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). A novel class of coronavirus SARS-CoV-2 causes deadly severe upper respiratory illnesses, the coronavirus disease 2019 (COVID-19), and many serious secondary health complications was first identified in 2019 . It is r++), whereas hemin contains ferric cation (Fe+++). In pharmacological frameworks, hemin is formulated with human albumin  to reduce the risk of phlebitis (or Venitis) and to enhance the stability of the active compound.HO-1 is a Nrf2-regulated gene that is activated by extracellular signaling and the HO-1 mRNA is translocated from nucleus to cytoplasm where it is translated into the functional HO-1 protein A. The inSince the discovery of HO-1 in 1968  as the cAn effective protection during early stage of viral exposure by restoring the viral-damaged host response is critical for our first defense against upper respiratory infections. In almost all viral infections, both pathogen and cellular gene transcription factors cooperatively promote active replication of the invaded pathogens. These factors contribute substantially from pathogen binding to the target cells and then internalization with subsequent replication, leading to the development of clinical symptoms followed by progression of the disease. HO-1 induction remarkably restricts replication of numerous viruses and thus is considered as a key mediator of the host defense mechanism.Interaction between viruses and host cell regulation is a key component in the initial response to infections and subsequent pathogenic events. Therefore, overcoming the viral-host defense evasion mechanism is pivotal to stimulate innate cellular immunity. Physiologically stimulated cellular HO-1, a widely distributed, inducible cytoprotective enzyme, presents a unique opportunity to enhance the normal host cell protective response. Failure of the immune system to defend against an invading pathogen poses serious challenges in managing the medical consequences ,25, partStimulation of the hemin-induced HO-1-dependent defense system along with mass vaccination might also facilitate immunity against constantly emerging pathogens than vaccines alone. This can be accomplished by substantially reducing intracellular viral loads and/or possibly by selective heme-induced toxicity of less HO-1-expressing SARS-infected cells. Because of high affinity of heme for albumin and hemopexin, it is not toxic to normal cells. Interventions involving activation of the powerful cellular HO-1-dependent cellular response by its natural substrate hemin could enable additional a safe viable option for reducing the severity of infections and disease progression. Therefore, HO-1-depenent strong resistance to various viruses for blocking or limiting their replication and associated pathogenesis should be explored to gain additional insights for mitigating SARS-CoV-2 infection. Scientific studies on evaluating the role of inducing HO-1 should provide an effective and productive outcome in advancing new therapeutic options.Modulation of the innate HO-1 may pave ways to provide new key insights into disrupting the replication cycle between the viruses and host factors for an alternative or concurrent effective physiological therapeutic strategy to effectively ameliorate infections by SARS-CoV-2 or other emerging viral pathogens. Activation of cellular HO-1-dependent cellular response could enable additional safe viable option for reducing SARS-CoV-2 infections, especially in the early stage of infection. In addition, since the activation of the innate immune activation also triggers antigen-specific adaptive immune responses , HO-1 acAlthough the approach for stimulated innate HO-1-dependent cellular protection presented in this Viewpoint can be generally applicable to most viral infections due to its capacity to enhance strong cellular resistance, it is particularly pertinent to the current problems of constantly emerging SARS-CoV-2 viral mutants. A number of identified SARS-CoV-2 variants are associated with reduced capacity of neutralization by antibodies generated against individual viral strains either naturally or produced by vaccination, in addition to increased transmissibility and pathogenesis . These sInnate cellular immunity is the first line of host defense mechanism against invading pathogens. The innate immune system plays an initial critical role in the clearance of viral infections. Furthermore, while the innate immune response is immediate, the secondary immune response is not generated until several days or weeks after infection. However, despite the powerful capacity of the initial innate host defense mechanism to resist all invading pathogens, it has not yet been adequately investigated as a form of initial protection against deadly viruses, such as SARS-CoV-2. Because HO-1 is widely distributed as an inducible intracellular enzyme in all cells, it presents a novel class of innate host defense system against deadly viruses. Therefore, pharmacological evaluation of HO-1-dependent strong cellular immunity may provide valuable insights into mitigating deadly viral infections, including SARS-CoV-2 mutant variants, and also for reducing their associated serious pathogenic consequences in a safe and effective manner. Physiologically induced innate HO-1 might also help facilitate the development of immunity against constantly emerging pathogens than vaccines alone by reducing viral loads and hence minimizing transmissions. Interventions involving activation of cellular HO-1-dependent cellular response could enable potentially safe viable options for reducing the severity of SARS-CoV-2 infections.Because COVID-19 is related to a marked dysregulation of the immunity by viral invasion of the innate immune system , emphasiCOVID-19 is an important area of an enormous current global public health concern. The HO-1-depenent resistance to viruses, viral replication and associated pathogenesis may provide additional medical modalities for SARS-CoV-2 that at present are of the highest priority. Therefore, future studies should also focus on the HO-1 regulatory pathways as viable therapeutic options for SARS-CoV-2 infections and in regulating innate host cellular response. Such studies, as stated above, could potentially enhance the effectiveness of the humoral immune response produced naturally in COVID-19 recovered patients or possibly in vaccines administered individuals.The host immune response to the SARS-CoV-2 causes an excessive inflammatory reaction called cytokine storm. The cytokine storm in many COVID-19 cases is associated with tissue damages. Most commonly organs that are largely affected by SARS-CoV-2 infection is the lung. In most cases, the lung dysfunction is short-term, causing acute respiratory distress syndrome ,33. HoweAlthough, high level of inflammation called multisystem inflammatory syndrome is a rare but serious condition associated with COVID-19, which not just affects the heart and lungs, but other body organs, including kidneys, brain, eyes, or gastrointestinal organs are also inflamed . These iCOVID-19 is an enormous current global public health concern. The concept of HO-1-depenent strong cellular resistance to a wide range of viruses should be considered to explore additional therapeutic options in managing SARS-CoV-2 infection. Because of its generic, non-specific protective role against several viral infections, regardless of the types of virus strain and mutant variants, pharmacological stimulation of HO-1 presents a potentially promising approach for SARS-CoV-2. Scientific studies on evaluating the role of HO-1 against SARS-CoV-2 may be expected to yield an effective and productive outcome in advancing new medical modalities, also as an alternative or concurrent therapeutic strategy for treating SARS-CoV-2 infections. Based on HO-1-dependent inhibition of numerous pathogens by hemin, the active component of an FDA-approved drug for an unrelated medical condition, it may further advance its therapeutic potential for mitigating SARS-CoV-2 infection and its associated inflammatory consequences with little or no adverse effects.


Metal-coordination-directed macrocyclic complexes, in which macrocyclic architectures are formed by metal-ligand coordination interactions, have emerged as attractive supramolecular scaffolds for the creation of materials for applications in biosensing and therapeutics. Despite recent progress, uncontrolled multicyclic cages and linear oligomers/polymers is the most likely outcome from metal-ligands assembly, representing a challenge to current synthetic methods. Herein we outlined the state-of-art synthetic approaches to the metal-coordination-directed macrocyclic complexes by using foldable ligands or through assembly of amphiphilic ligands. This mini-review offers a guideline for the efficient preparation of metal-coordination-directed macrocyclic complexes with predictable and controllable structures, which may find applications in many biology-related areas. The fir12 . A varie12 . By cont12 . The resDespite the synthetic difficulty, metal-coordination-directed macrocyclic complexes have attracted the attentions of supramolecular chemists, owing to their pre-organized cyclic binding cavity where multiple binding sites enhance binding affinity and selectivity for a guest species . In thisi.e., pyridine motifs, at both the C- and N-termini. Upon Ag+ coordination, 7-/8-crossion knot or helical complexes can be generated, which further self-assemble into larger structures  specifically and have little side effects on normal cells, whereas its enantiomer NiM is inactive (NiP exhibit higher selectivity toward telomere G-quadruplex DNA (G4-DNA) in breast CSCs than NiM. Besides, the cationic Ni2+ centers in the metal-coordination complexes can bind to telomere G4-DNA via electrostatic interaction. The preferential interaction between NiP and G4-DNA resulted in the dissociation of telomere associated protein from telomeres, the damaging of telomere DNA and cancer cells apoptosis. The study demonstrates that the ability of chiral metal-coordination-directed macrocyclic complexes to function as chiral drugs for tumor treatment.Intriguingly, based on the same approach that utilizes angled aromatic structure of pyridine groups as ligands, Qu and co-workers also developed chiral metal-coordination-directed macrocyclic complexes inactive    Mishra . The phoThe synthetic approach of foldable or folded conformation of ligands is favored to guide the construction of various kinds of metal-coordinated-directed complexes. The conformation of ligands is significant to the cyclization, such as ligands with flexible and fine-tuning structure, the difference of coordination sites on ligands, as well as the bended structure of aromatic ligands that modified with pyridines. This approach imposes strict requirement on the structure of ligand, that the foldable conformation of ligands should feature a certain angularity to promote cyclization. However, for linear ligands, they would tend to form linear oligomers/polymers in the presence of metal ions. It means that we should develop the other approaches to favor the formation of metal-coordination-directed macrocyclic complexes between metal ions and flexible ligands.Inspired by self-assembly of amphiphilic surfactants forming micelles and vesicles, supramolecular amphiphiles formed by metal-coordination interactions have been developed to afford a novel approach to build metal-coordination-directed macrocyclic complexes, wherein the ligands can be flexible.in-situ formed Ag+-thiol linear coordination polymers by employing the cysteine derivatives as thiol ligands  and electrostatic interaction, π-π interactions between side-chains of ligands could also facilitate the formation of the coordination polymers. Recently, Jiang et al.  and hydrophobic n-butylmercaptan (n-BuSH) formed Ag+-thiol coordination polymers, the absorption band at 285 nm demonstrated that Ag+ on the backbone linked by localized Ag+···Ag+ interaction. Cys and n-BuSH served as building blocks located on the outward and inward of complexes backbone, respectively. By virtue of the hydrophilic-hydrophobic interactions and Ag+···Ag+ interaction, the contrivable ligand as block unit and metal ion directed the formation of macrocyclic complexes. For most of supramolecular polymers, they display majority rules. Intriguingly, in this work, the chirality of Cys ligand is transferred to the macrocyclic backbone, and the metal-coordination-directed complexes exhibit an unusual anti-Z-shaped CD-ee dependence, previously termed as a “racemate rules effect”  measurements demonstrated that the metal complexes formed in the presence of glucose are more monodisperse than those formed by other monosaccharides of similar structures. The synthetic strategy of supramolecular amphiphiles in-situ triggered by metal ions is applicable to constructing metal-coordination-directed macrocyclic complexes, which also can be used for recognizing biomolecules such as glucose.Based on the strategy of co-assembling hydrophilic and hydrophobic ligands into the Agg et al.  employedg et al. , whereinin-situ formed metal-coordination-directed macrocyclic complexes are easy to obtain and show unique merits. However, it is of grand challenge to obtain macrocyclic complexes of precisely uniform size. There are also difficulties in structural characterizations as the complexes can conglomerate in drying process.Comparing with the complex synthetic strategies of macrocyclic compounds, We summarized two types of metal-coordination-directed macrocyclic complexes that have been synthesized efficiently based on smart ligand choices. 1) A variety of macrocycles were described using the carefully designed ligands with folded structures that geometrically satisfy desired ring topology. Many of the resultant macrocycles have found applications in biology-related areas, including biomolecular recognition, drug delivery, and therapeutics for AD and cancer. 2) Cyclic metal-ligand arrays generated by exploiting micellar and vesicular assembly of flexible amphiphilic ligands in the presence of metal ions.There are significant opportunities and challenges in the area of metal-coordination-directed macrocyclic complexes. To achieve real-world applications such as clinical diagnosis and therapeutics, a key future research direction is to develop easily synthesized systems that can be derivatized with functional units such as biomolecule recognition sites and optical sensing units. In the design of macrocyclic metal complexes, the cooperative effect of multiple interactions, such as π-π interactions, metal-metal interactions and hydrogen bonding have been proved to benefit the construction of metal-coordination-directed macrocyclic complexes. In this context, other interactions, such as halogen and chalcogen bonds, are less exploited and could be alternative driving forces to promote the synthesis of metal-coordination-directed macrocyclic complexes. The excellent properties of metal-coordination-directed macrocyclic complexes, for example, flexible and easy tuning structure create a special microenvironment to bind guest molecules and many functional modification sites have drawn significant attentions, leading to applications in biological field, such as molecular recognition and therapeutics. We anticipate this mini-review will facilitate the synthesis of metal-coordination-directed macrocyclic complexes with diverse structures and functions.


Studies have demonstrated the efficacy of mechanical devices at delivering high-quality cardiopulmonary resuscitation (HQ-CPR) in various transport settings. Herein, this study investigates the efficacy of manual and mechanical HQ-CPR delivery on a fire rescue boat.A total of 15 active firefighter-paramedics were recruited for a prospective manikin-based trial. Each paramedic performed two minutes manual compression-only CPR while navigating on a river-based fire rescue boat. The boat was piloted in either a stable linear manner or dynamic S-turn manner to simulate obstacle avoidance. For each session of manual HQ-CPR, a session of mechanical HQ-CPR was also performed with a LUCAS 3 . A total of 60 sessions were completed. Parameters recorded included compression fraction (CF) and the percentage of compressions with correct depth >5cm (D%), correct rate 100-120 (R%), full release (FR%), and correct hand position (HP%). A composite HQ-CPR score was calculated as follows: ((D% + R% + FR% + HP%)/4) * CF%). Differences in magnitude of change seen in stable versus dynamic navigation within study conditions were evaluated with a Z-score calculation. Difficulty of HQ-CPR delivery was assessed utilizing the Borg Rating of Perceived Exertion Scale.Participants were mostly male and had a median experience of 20 years. Manual HQ-CPR delivered during stable navigation out-performed manual HQ-CPR delivered during dynamic navigation for composite score and trended towards superiority for FR% and R%. There was no difference seen for any measured variable when comparing mechanical HQ-CPR delivered during stable navigation versus dynamic navigation. Mechanical HQ-CPR out-performed manual HQ-CPR during both stable and dynamic navigation in terms of composite score, FR%, and R%. Z-score calculation demonstrated that manual HQ-CPR delivery was significantly more affected by drive style than mechanical HQ-CPR delivery in terms of composite HQ-CPR score and trended towards significance for FR% and R%. Borg Rating of Perceived Exertion was higher for manual CPR delivered during dynamic sessions than for stable sessions.Mechanical HQ-CPR delivery is superior to manual HQ-CPR delivery during both stable and dynamic riverine navigation. Whereas manual HQ-CPR delivery was worse during dynamic transportation conditions compared to stable transport conditions, mechanical HQ-CPR delivery was unaffected by drive style. This suggests the utility of routine use of mechanical HQ-CPR devices in the riverine patient transport setting. Along with rapid cardiac defibrillation in shockable rhythms, rapid administration of high-quality cardiopulmonary resuscitation (HQ-CPR) improves outcomes in OHCA. According to the American Heart Association , HQ-CPR compressions necessitate a minimization of interruptions, a rate of 100-120, a depth >5 cm, and an adequate chest recoil. Mechanical CPR devices have been proposed as alternatives to manual CPR as a way to deliver reliable HQ-CPR while utilizing fewer human resources and preventing in-transport provider injury. Mechanical CPR devices have also been suggested to be efficacious in various transportation modalities, including ambulance, helicopter, and snow sled patient transport.Out-of-hospital cardiac arrest (OHCA) affects millions of people every year globally with high rates of mortality and long-term morbidity. Importantly in these studies, the boats were piloted in a linear course without turning. In real-life scenarios, HQ-CPR on a moving waterway vessel is likely affected by the vessel’s characteristics as well as the various forces that influence vessel displacement such as wind, waterway conditions, and waterway traffic. The effect of this displacement on the efficacy of manual HQ-CPR delivery has not been investigated. Additionally, the efficacy of mechanical delivery of HQ-CPR in the riverine rescue setting has not been investigated. As mechanical CPR machines are strapped to the patient, they may yield more consistent results that are less perturbed by the displacement experienced by a watercraft in transit.In simulation studies, HQ-CPR performance is negatively affected by boat speed and ocean conditions in trained and lay personnel, causing up to a 30% decrease in effective compression delivery.In this study, the authors’ aimed to accomplish two goals. First, they sought to evaluate the effect of navigation style on HQ-CPR delivery by both human providers and a mechanical CPR device while aboard a moving small inland watercraft. Second, they sought to directly compare HQ-CPR delivered manually by human providers to HQ-CPR delivered by a mechanical device under these differing navigation conditions.After receiving an exemption from the UC Davis Institutional Review Board Administration , active firefighter-paramedics with water-based rescue experience were recruited from local agencies for a prospective manikin-based trial. Participant demographic information that was recorded included age, weight, height, sex, and years of paramedic experience. The vessel was operated at a target velocity of approximately 13.4 meters/second (30 miles/hour) in either a stable linear manner or dynamic S-turn manner to simulate obstacle avoidance  while navigating on a 22-foot rigid hull inflatable rescue boat . Parameters recorded included compression fraction (CF%) and the percentage of compressions with correct depth >5cm (D%), correct rate 100-120 (R%), full release (FR%), and correct hand position (HP%). A composite HQ-CPR score was adapted from previous literature to incorporate all Level 1 or Level 2a recommendations from the AHA and was calculated as follows: ((D% + R% + FR% + HP%)/4) * CF%).Mean, median, standard deviation, and interquartile ranges of HQ-CPR parameters were calculated for each study condition. A two tailed paired t-test as well as a change score and 95% confidence interval were calculated to evaluate for statistical differences in HQ-CPR outcomes between the manual stable and manual dynamic study conditions. A two tailed heteroscedastic t-test and 95% confidence intervals were calculated to evaluate for statistical differences in HQ-CPR outcomes between the mechanical stable versus mechanical dynamic conditions, manual stable versus mechanical stable conditions, and manual dynamic versus mechanical dynamic conditions. A Z-score was calculated to evaluate the difference in the magnitude of change seen in the manual stable versus manual dynamic conditions compared to the change seen in the mechanical stable versus mechanical dynamic conditions. Difficulty of manual HQ-CPR delivery during stable and dynamic sessions was compared using a paired two tailed student’s t-test.th, 75th percentile: 7.5, 23) years of Emergency Medical Service experience.A total of 15 participants were recruited for this study. All participants were active-duty firefighter-paramedics with Advanced Cardiac Life Support/ACLS certification. Participant demographics can be seen in Table The HQ-CPR results from the four study conditions can be seen in Table Z-score calculations can be seen in Table Borg Rating of Perceived Exertion among participants was significantly higher for manual HQ-CPR delivery during dynamic navigation compared to manual HQ-CPR delivery during stable navigation . When asked to compare the difficulty of HQ-CPR delivery in a small waterway vessel during stable navigation with performing HQ-CPR in a moving ambulance, eight (53%) participants responded that it was the same difficulty level and four (27%) participants responded that it was somewhat more difficult or much more difficult. When asked to compare the difficulty of HQ-CPR delivery in a small waterway vessel during dynamic navigation to performing HQ-CPR in a moving ambulance, two (13%) participants responded that it was the same difficulty level and thirteen (87%) participants responded that it was somewhat more difficult or much more difficult. Though this study was performed during calm weather conditions, the vessel was piloted in two separate patterns: a stable pattern to simulate transportation in calm, unobstructed waterways as well as in a dynamic S-turn pattern to simulate avoidance of obstacles and boat displacement during more difficult conditions. Examples of obstacles that may be encountered during riverine patient transport include recreational boaters, kayakers, paddle boarders, swimmers, floating debris, or static geologic and botanical features.This study investigated the effectiveness of manual and mechanical delivery of HQ-CPR chest compressions in a small waterway rescue vessel. Navigation and the delivery of HQ-CPR during transport in a riverine setting differs dramatically from land-based transport due to differences that include vehicle structure, braking systems, transportation medium , and the presence of an underlying current. Previous studies have demonstrated that increasing vessel speed, rough waters, and provider fatigue contributed to poor HQ-CPR outcomes during maritime transport.In this study, manual HQ-CPR delivered during stable navigation was significantly better than manual HQ-CPR delivered during dynamic navigation. In particular, FR% and R% trended negatively in the dynamic arm, though did not reach statistical significance. These results likely reflect an increase in the amount of leaning required to maintain balance during navigation. The off-balancing forces experienced during dynamic navigation were likely higher than those experienced during stable navigation, requiring the use of intermittent tripoding by the participants. This likely reduced the amount of full chest recoil experienced by the manikin as well as altered the delivery rate of chest compressions. The use of a metronome has been shown to improve compression rate during the delivery of HQ-CPR, however noise pollution from the wind and boat engine made metronome use in this study impractical. In the current study, dynamic navigation resulted in a higher rating of perceived exertion and likely contributed to the lower HQ-CPR scores. Importantly, HQ-CPR in this study was delivered with the providers in the customary water rescue kneeling position. This has been shown to improve the quality of HQ-CPR delivery and may have served to reduce but not eliminate the effects of postural fatigue. Despite this fact, participants still found HQ-CPR delivery during dynamic navigation to be more difficult than their standard HQ-CPR delivery in a moving ambulance. Additionally, the participants in this study were firefighter-paramedics with rigorous physical fitness standards. Thus, the rating of perceived exertion during dynamic navigation may be higher in participant populations with lesser physical conditioning.Previous studies have suggested that fatigue during maritime HQ-CPR delivery is largely due to local fatigue of postural muscles rather than aerobic metabolic demand.In contrast to the manual HQ-CPR delivery, the efficacy of mechanical HQ-CPR delivery was unaffected by navigation style. There was no significant difference in any of the recorded HQ-CPR parameters seen between the stable mechanical HQ-CPR and dynamic mechanical HQ-CPR study conditions. Additionally, Z-score evaluation demonstrated significantly less change in composite HQ-CPR score during different navigation styles for mechanical HQ-CPR delivery compared to manual HQ-CPR delivery and trended towards significance for FR% and R%. This suggests that mechanical HQ-CPR delivery is much less affected by increased off-balancing forces and could be valuable when it is necessary to deliver HQ-CPR in scenarios that increase displacement of the vessel during transport. Mechanical HQ-CPR also resulted in a lower average compression rate with significantly less variability for both stable and dynamic evolutions. For both stable and dynamic navigation, manual HQ-CPR had a duty cycle of approximately 39.4%, which is similar to previous studies. In contrast, mechanical HQ-CPR achieved a duty cycle of approximately 53%, closer to the 50% goal recommended by the AHA, although this is only a Level 2b recommendation with Level C quality of evidence.Mechanical HQ-CPR out-performed manual HQ-CPR in terms of FR%, R%, and composite HQ-CPR score for stable and dynamic evolutions and trended towards better HP% for both types of drive style. Though there was no difference in D% between manual and mechanical HQ-CPR for stable or dynamic navigation, mechanical HQ-CPR averaged a shallower depth than manual HQ-CPR, achieving a mean depth within the 5-6cm depth range suggested by the AHA. It may be that a more targeted approach to the use of mechanical HQ-CPR devices would yield superior results rather than universal deployment. Scenarios that limit provider access to the patient such as helicopter transport or prolonged gurney transport in narrow passages have been suggested to be more appropriate for mechanical HQ-CPR use. Like helicopters, small waterway vessels afford only restricted space for providers to operate, limiting the speed and reliability with which procedures, including HQ-CPR, can be performed. Transportation in a small water rescue vessel also necessitates multiple episodes of relocation, such as from the initial contact point into the vessel, from the vessel into an ambulance, and from the ambulance to the final point of care. Future research investigating HQ-CPR delivery during a prolonged transport and relocation periods within the riverine setting may further elucidate the benefits of mechanical HQ-CPR delivery. A factor of riverine patient transport that may negatively affect the efficacy of mechanical HQ-CPR is moisture on the patient’s chest, which may increase slippage of the device during compression cycles.Despite its demonstrated effectiveness in a variety of simulated scenarios, mechanical HQ-CPR has yet to demonstrate a benefit over manual HQ-CPR in prospective randomized trials in terms of patient outcomes.Limitations include the limited number of evolutions performed, the omission of ventilations, omission of rhythm checks, a manikin-based approach rather than real-life scenarios, and the performance of only two minutes of CPR, which would likely mitigate fatigue factor and hands-off time. This study also failed to evaluate the effects of patient movement into or out of the rescue vessel and did not account for moisture which may be present in a river-based rescue scenario.This study suggests the superiority of mechanical HQ-CPR delivery over manual HQ-CPR delivery during patient transport in a riverine setting. Whereas manual HQ-CPR delivery was negatively affected during dynamic transportation conditions, mechanical HQ-CPR delivery was unaffected. This suggests the utility of routine use of mechanical HQ-CPR devices in the riverine patient transport setting, though further investigation is necessary.


Deep learning enables bypassing the tradeoffs between imaging speed, field of view, and spatial resolution in autofluorescence-harmonic microscopy. Two-photon autofluorescence (2PA) of flavin adenine dinucleotide (FAD) and three-photon autofluorescence (3PA) of nicotinamide adenine dinucleotide (NADH) permit noninvasive monitoring of metabolic activities2. Second-harmonic generation (SHG) microscopy provides morphological and functional characterization of anisotropic biological structures, such as collagen3, muscle4, and microtubules5. Third harmonic generation (THG) microscopy enables elucidating on cellular and tissue organizations by probing intra- and extracellular membranes, and extracellular matrix structures6. Recent advances in simultaneous label-free autofluorescence- multiharmonic (SLAM) microscopy further expands the nonlinear optical microscopy’s utility in intravital imaging7 and slide-free, stain-free histopathology9.Label-free nonlinear optical microscopy is an emerging technique for probing biological structures and functions without exogenous labels or dyes. Thus, it provides an attractive solution to gain biological insights without perturbing the native states of biological samples and processes. For example, vibrational spectroscopic imaging techniques10, a novel deep learning augmented microscopy framework is developed to overcome the physical tradeoffs. The proposed “deep learning autofluorescence-harmonic microscopy” (DLAM) is demonstrated on human pathological tissues for multimodal imaging, including 2PA of FAD, SHG and 3PA of NADH, with enhanced spatial resolution and much reduced acquisition time. This advancement may find broad utilities in the studies of biology and neuroscience.Despite these advances, the implementations of all nonlinear optical microscopy techniques rely on laser scanning. This makes it challenging to simultaneously achieve a wide field of view (FOV), high spatial resolution, and fast imaging speed with sufficient signal-to-noise ratio (SNR) in the measurement. In this work by Shen et al.11, which overcome different aspects of physical limitations by combining novel instrumentation and deep learning. For example, strategies have been developed to first acquire low-SNR images at high speed and low light exposure and later enhance the SNR by deep learning to alleviate photo-damages in fluorescence microscopy12 and SRS microscopy13. Deep learning-based super-resolution reconstruction has been demonstrated to bypass the limitation of FOV14. Data-efficient acquisition schemes by deep learning have been developed for multi-shot quantitative imaging techniques, such as Fourier ptychographic microscopy15, single molecule localization microscopy16, and structured illumination microscopy17. We envision this deep learning augmented approach may fundamentally push the imaging limits and ultimately revolutionize the field of microscopy.Broadly speaking, DLAM adds to the rapid-growing list of deep learning augmented microscopy techniques


Editing events from RNA-editinghigh pattern were significantly associated with glutamate receptors and regulated synaptic transmission. Interestingly, most non-synonymous high-editing sites were mapped to ion channel genes of glutamatergic synapse which were associated with neurotransmission by controlling ion channel permeability and affecting exocytosis. Meanwhile, these non-synonymous editing sites were evolutionarily conserved and exhibited a consistently increasing editing levels between mouse and human retinal development. Single-cell RNA-seq data analysis revealed that RNA editing events prefer to occur in two main cell types including bipolar and amacrine cells. Genes with non-synonymous high-editing sites were enriched in both bipolar cells and retina ganglion cells, which may mediate retina ganglion cell differentiation by altering channel ion permeability. Together, our results provide novel insights into mechanism of post-transcriptional regulation during retinal development and help to develop novel RNA editing-guided therapeutic strategies for retinal disorders.Adenosine-to-inosine (A-to-I) RNA editing leads to functional change of neurotransmitter receptor which is essential for neurotransmission and normal neuronal development. As a highly accessible part of central nervous system, retina has been extensively studied, however, it remains largely unknown how RNA editing regulates its development. Here, a genome-wide screening of high-confidence RNA editing events were performed to decipher the dynamic transcriptome regulation by RNA editing during mouse retinal development. 2000 high-confidence editing sites across eight developmental stages of retina were called. Three unique patterns (RNA-editing Due to structural similarities, inosines (I) are read by the cellular machinery as guanosine (G) Transcriptional profiling of genome-wide RNA sequencing confirmed a large number of RNA editing sites in many important neuronally expressed genes As the most accessible part of the central nerve system(CNS), neural retina is an excellent model system to analyze key aspects of neurogenesis As the most accessible part of CNS, the mechanisms underlying retinal development are highly conserved across vertebrates, making it an excellent model system for studying nerves. Although RNA editing is essential for the functions of nervous system, their dynamic changes and specific roles in retinal development and mechanisms are unclear.high pattern, RNA-editingmedium pattern and RNA-editinglow pattern were identified. Among them, we found that RNA-editinghigh pattern showed a clearly increasing trend during retinal development. The associated genes are significantly enriched in glutamatergic genes, which play a crucial role in neurotransmission by controlling ion channel permeability and affecting exocytosis. Moreover, non-synonymous RNA editing sites in glutamate ion receptors are associated with bipolar cells and retinal ganglion cells. All in all, our study provided a genome-wide map of RNA editing events and characterized different RNA-editing-mediated post-transcriptional modification patterns during retinal development. The novel high-confidence retina-specific editing sites expanded the known RNA editing resource.In this study, we developed a framework to comprehensively characterize dynamic RNA editing events during mouse retinal development. Three distinct patterns including RNA-editing22.1https://www.ncbi.nlm.nih.gov/sra, accession number SRP090043) The bulk RNA-seq data for mouse retina development were downloaded from the NCBI Sequence Read Archive (2.2We used a pipeline adapted from a previously reported method 2.3https://genome.ucsc.edu/).To further analyze the function of RNA editing sites, we used ANNOVAR 2.4https://github.com/YuLab-SMU/clusterProfiler). We used all mouse genes as a reference gene set to compare with edited genes for functional enrichment analysis. Only the terms of "biological process" were selected for GO enrichment analysis. Enrichment with FDR was adjusted by Benjamini-Hochberg multiple comparisons. The selected KEGG pathways related to RNA editing were visualized using the Pathview package We performed KEGG pathway and Gene ontology (GO) enrichment analyses by the enrichGO and enrichKEGG function in the R package clusterProfiler 2.5https://meme-suite.org/tools/meme) Motif prediction was performed using MEME-ChIP in the MEME suit  deletion upstream of the editing site and some G enrichment downstream A. Moreovi.e. in most species, a large proportion of editing events occur in repeating elements may form long double-stranded RNA structures that are more favorable for enzyme binding To examine the genome distribution of the RNA editing sites, we annotated the sites by ANOVAR tool To explore possible functional significance of these RNA editing events across retinal development, we performed GO functional enrichment analysis. The edited genes  containing at least one high-confidence RNA editing site were found to be significantly enriched in retinal development in the camera-type eye, dendrite development and glutamate receptor signaling pathways (p.adjust < 0.05) F. FunctiTo sum up, we identified 2000 high-confidence RNA editing sites across eight retinal development stages. More than third of them were retina-specific, which may have some characteristics different from that of brain although they were all enriched in neurons.3.2To further characterize these editing sites, we examined the number of high-confidence editing sites located in major repeat families in mouse. On the one hand, consistent with human beings, high-confidence RNA editing sites were primarily located in short interspersed element (SINE) (75.9%) A that weTo further dissect RNA editing events in the B1 element, editing levels between repeat family regions and non-repeat regions were compared. Result demonstrated that RNA editing level of B1 elements was significantly higher  than other repeat elements and non-repeat regions during retinal development, while there was no editing level difference between other repeat elements and non-repeat regions B.Altogether, during mouse retinal development, high RNA editing events prefer to occur at B1 repeat elements, which may form dsRNA structure that allows ADAR enzymes to bind easily to mRNA sequences 3.3To explore changes of RNA editing during retinal development, we quantified genome-wide editing degree by calculating A-to-I RNA editing level across all stages. For high-confidence RNA editing sites, editing rate was defined as the ratio of reads number that supported adenosine (A) to guanosine (G) to all reads number covered the sites(G/(G + A)). We observed that the RNA editing level gradually increased during retinal development A, which high; green, RNA-editingmedium; blue, RNA-editinglow pattern)  A. We chopattern)  B as thehigh pattern sites were highly enriched in glutamate receptor and regulated synaptic transmission. In contrast, RNA-editinglow pattern editing sites were significantly enriched in essential functions, such as mRNA splicing and binding-related genes  and with highest proportion of non-synonymous editing sites among all patterns during retinal development (high pattern and RNA-editingmedium pattern) had a higher proportion of editing sites in the repeat region than the RNA-editinglow pattern  were mapped to ion channel proteins (Cacna1d) and glutamate ionotropic receptors   A. Among high pattern also showed increasing trend with higher editing levels compared with common sites (high pattern during retinal development.Interestingly, six of the eight non-synonymous editing sites were evolutionarily conserved between mouse and humans B. Duringon sites C. Moreovon sites . These r3.5The major cell types of retina has been well-characterized and showed a stereotyped birth order during development To further explore the contribution of each retinal cell type to RNA editing events during retinal development, gene expression and RNA editing profiles of different retinal major cell types were analyzed. The enrichment scores of edited genes were calculated to in bulk-seq for each individual cell using the Addmodulesocre function of Seurat package. As indicated by the boxplot, amacrine cells possessed highest proportion of edited genes A and B. To investigate the specificity of these non-synonymous high-editing sites in various cell types, enrichment scores of these non-synonymous edited genes for each cell were calculated. We found that these non-synonymous edited genes were enriched in amacrine bipolar cells, retinal ganglion cells, HC cells and amacrine cells D, in whi4high pattern were significantly enriched in glutamatergic genes and ion channel genes, which is consistent with previous findings that high editing sites were mainly detected in neurons low patterns were observed in genes involved in RNA binding and splicing. As reported, RNA binding proteins such as FMRP and ILF3 could directly regulate RNA editing by affecting the interaction between ADAR enzyme and RNA sequence cis-acting signals that regulate splicing activity and destabilize double-stranded structures To our best knowledge, this is the first study with comprehensive characterization of A-to-I RNA editing events in mouse retina. In addition to identification of novel high confidence retina-specific editing sites, we featured distinct dynamic post-transcriptional regulation patterns during retinal development. Edited genes from RNA-editingIn humans, most RNA editing events were found in transcribed repeat elements high patterns in retinal development, play a crucial role in neurotransmission by controlling ion channel permeability and affecting exocytosis Non-synonymous mutations were largely considered deleterious because they resulted in the change of amino acid sequence. However, these non-synonymous editing sites, from RNA-editingComparative analysis of vertebrate and invertebrate genomes suggests that a substantial proportion of vertebrate genes had no homologous genes in the invertebrate genome During our investigation, we suffered from short reads, low coverage and easy mismatches when using single-cell data for RNA editing analysis. To address these issues, we merged reads from the same cell types to increase coverage and used RNA editing profiles from bulk-seq as background to avoid false sites generated by mismatches. With the development of single-cell sequencing technology, we may solve these problems possibly at the sequencing level and identify more cell-specific editing sites. Single-cell analysis of RNA editing found that non-synonymous editing was associated with bipolar cells and retinal ganglion cells. Consistent with previous reports, our results revealed that non-synonymous RNA editing could modulate the electrical signaling pathway between bipolar cells and retinal ganglion cells by affecting glutamate ion receptor permeability during stimulus transmission.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


The global coronavirus disease 2019 (COVID-19) pandemic is currently ongoing. It is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A high proportion of COVID-19 patients exhibit gastrointestinal manifestations such as diarrhea, nausea, or vomiting. Moreover, the respiratory and gastrointestinal tracts are the primary habitats of human microbiota and targets for SARS-CoV-2 infection as they express angiotensin-converting enzyme-2 (ACE2) and transmembrane protease serine 2 (TMPRSS2) at high levels. There is accumulating evidence that the microbiota are significantly altered in patients with COVID-19 and post-acute COVID-19 syndrome (PACS). Microbiota are powerful immunomodulatory factors in various human diseases, such as diabetes, obesity, cancers, ulcerative colitis, Crohn’s disease, and certain viral infections. In the present review, we explore the associations between host microbiota and COVID-19 in terms of their clinical relevance. Microbiota-derived metabolites or components are the main mediators of microbiota-host interactions that influence host immunity. Hence, we discuss the potential mechanisms by which microbiota-derived metabolites or components modulate the host immune responses to SARS-CoV-2 infection. Finally, we review and discuss a variety of possible microbiota-based prophylaxes and therapies for COVID-19 and PACS, including fecal microbiota transplantation (FMT), probiotics, prebiotics, microbiota-derived metabolites, and engineered symbiotic bacteria. This treatment strategy could modulate host microbiota and mitigate virus-induced inflammation. Microbiota serve multiple important functions in and on the human body such the decomposition of indigestible carbohydrates and proteins, nutrient digestion and absorption, vitamin biosynthesis, and host immunity induction, instruction, and function.13 The microbiota influence human health and are associated with several diseases.There are tenfold more bacterial cells in the human microbiota than there are human tissue cells and there are 100-fold more bacterial than human genes.14 Patients with COVID-19 present with symptoms of respiratory infection including fever, fatigue, abnormal chest X-ray, cough, and shortness of breath.18 Furthermore, a high proportion of COVID-19 patients also exhibit gastrointestinal manifestations such as diarrhea, nausea or vomiting, anorexia, and abdominal pain  pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has posed serious threats to public health and the global economy.The aim of this review was to summarize the relationships between microbiota and COVID-19 in terms of their clinical relevance and immunological mechanisms. We also explored various interventions that target microbiota, are based on the immunological interplay between the microbiota and COVID-19, and could optimize anti-SARS-CoV-2 therapies.Betacoronavirus.31 It encodes membrane (M), nucleocapsid (N), spike (S), and envelope (E) structural proteins and multiple non-structural proteins.32 SARS-CoV-2 obligately requires the S protein to penetrate host cells.33 On the virion, the S protein is a homotrimer comprising S1 and S2 subunits. The former binds host angiotensin-converting enzyme-2 (ACE2) while the latter mediates membrane fusion.36 The virus hijacks host cell-surface proteases such as transmembrane protease serine 2 (TMPRSS2) which, in turn, activates viral S protein, cleaves ACE2 receptors, and facilitates viral binding to the host cell membrane.39 In addition to ACE2 and TMPRSS2-mediated entry, SARS-Cov-2 can also utilize the phagocytosis or endocytosis function of host cells to invade certain immune cell types such as macrophages.40 ACE2 and TMPRSS2 are strongly expressed in the respiratory and gastrointestinal tracts. As the latter communicates with the external environment, they are the major targets of SARS-CoV-2 invasion  occur in healthy upper airways including the nasal cavity, nasopharynx, and oropharynx. In contrast, the lung has slightly lower bacterial densities (~102 U−1).48 Healthy upper airways are typically populated by Staphylococcus, Propionibacterium, Leptotrichia, Rothia, Dolosigranulum, Haemophilus, Moraxella, Veillonella, and Corynebacterium. Veillonella, Fusobacterium, and Haemophilus are the main genera inhabiting healthy lungs. Prevotella and Streptococcus occur in both upper airways and lungs.50 Evidence from prior research demonstrated that commensal bacteria in the respiratory tract help prevent pathogens from establishing infections and spreading on the mucosal surfaces.51 This phenomenon is known as “colonization resistance”. Hence, respiratory tract microbiota might help prevent SARS-CoV-2 infection. By preventing SARS-CoV-2 colonization on the mucosal surfaces, microbiota could inhibit the virus infection to a certain degree.Gas exchange is the primary function of the respiratory tract. To perform gas exchange efficiently, adult human airways have approximately 40-fold larger surface area than skin.52 As ACE2 and TMPRSS2 are highly expressed in the gastrointestinal tract, SARS-CoV-2 also targets the gut.46 Several studies reported that stool samples from patients with COVID-19 were positive for SARS-CoV-2 viral RNA. Endoscopy revealed colon damage in these patients. Thus, SARS-CoV-2 can infect the gastrointestinal tract.58 A population-based study conducted in China showed that viral RNA was detected in the stool samples of ≤53% of all COVID-19 patients.59 A biopsy performed on a COVID-19 patient disclosed the SARS-CoV-2 protein coat in the stomach, duodenum, and rectum.59 Therefore, both SARS-CoV-2 and its close relative SARS-CoV can infect the gut. The gut microflora are more abundant and diverse than those in the respiratory tract.60 A few studies confirmed that gut microbiota help regulate intestinal immune homeostasis and pathogen infection.63 For this reason, gut bacteria may be vital to the host immune response to SARS-CoV-2 infection.Respiratory droplet and fomite transmission may be the primary modes of SARS-CoV-2 transmission. Nevertheless, a recent study suggested that SARS-CoV-2 may also be spread via the fecal–oral route.64 The incubation period is usually ~1–14 d. In most cases, though, it is 3–7 d.16 Approximately 97.5% of all COVID-19 patients develop symptoms within 14 d of infection. Only 2.5% of them remain asymptomatic.16 The clinical manifestations of COVID-19 are highly variable but commonly include shortness of breath (53–80%), sputum production (34.3%), dry cough (60–86%), and sore throat (13.9%).19The disease course of COVID-19 is characterized by the incubation, symptomatic, hyperinflammation, and resolution periods.81 A study conducted in Chile reported that out of 7,016 patients with COVID-19, 11% displayed gastrointestinal symptoms.74 Jin et al. reported that among 651 patients with COVID-19 in Zhejiang, China, 8.6% exhibited diarrhea while 4.15% presented with nausea or vomiting.21 Gastrointestinal symptoms are associated with a relatively higher risk of hospitalization and/or greater disease severity. In severe and/or critical patients, the disease progresses and causes complications such as acute respiratory distress syndrome (ARDS), sepsis, secondary pathogen pneumonia and end-stage organ failure. As microbiota maintain respiratory and gastrointestinal homeostasis and health, the foregoing COVID-19-associated symptoms may link microbiota with SARS-CoV-2 infection.Several clinical studies reported that 11–39% of all COVID-19 patients have gastrointestinal symptoms, including nausea, vomiting, diarrhea, and abdominal pain  of all severe COVID-19 patients but not in any mild cases. The major respiratory microbial taxa in the critically ill COVID-19 patients consisted of Burkholderia cepacia complex (BCC), Staphylococcus epidermidis, and/or Mycoplasma spp. In 23.1% (3/13) of all severe COVID-19 cases, clinical sputum and/or nasal secretion cultures confirmed the presence of BCC and S. epidermidis. In a critical COVID-19 patient, there was a time-dependent secondary Burkholderia cenocepacia infection and expression of multiple virulence genes that might have accelerated disease progress and hastened eventual death. A study conducted at Huashan Hospital in Shanghai, China reported that among 62 COVID-19 and 125 non-COVID-19 pneumonia cases, potentially pathogenic microbes were detected in 47% of the former, and 58% of the pathogens were respiratory viruses.24 A recent study demonstrated a link between respiratory microbiota and COVID-19 disease severity.23 Several potential confounding factors contributed to microbiota alteration in COVID-19. These included time spent in the intensive care unit (ICU), antibiotic administration, and type of oxygen support. The authors integrated microbiome sequencing, viral load determination, and immunoprofiling, and identified specific oral bacteria associated with relatively higher levels of proinflammatory markers in COVID-19 patients.Emerging evidence suggests that the microbiota of the respiratory and gastrointestinal tracts are dramatically altered in COVID-19 patients. An early study in Guangdong Province, China revealed that the respiratory microbiota in COVID-19 patients have reduced α-diversity and elevated levels of opportunistic pathogenic bacteria.26 The researchers showed that compared with the gut microbiomes of healthy persons, those of patients with COVID-19 had low abundances of the anti-inflammatory bacteria Lachnospiraceae, Roseburia, Eubacterium, and Faecalibacterium prausnitzii. The feces of COVID-19 patients were enriched in opportunistic pathogens known to cause bacteremia such as Clostridium hathewayi, Enterobacteriaceae, Enterococcus, Actinomyces viscosus, and Bacteroides nordii. Gut dysbiosis persists even after clearance of SARS-CoV-2 infection or recovery from it. The gut fungi and virome comprise parts of the gut microbiota and are also altered in response to SARS-CoV-2 infection. Another study observed relatively increased proportions of opportunistic fungal pathogens such as Candida albicans, C. auris, and Aspergillus flavus in the feces of COVID-19 patients.25 Previous investigations showed that the foregoing fungal pathogens are associated with pneumonia and other respiratory infections.85 Therefore, gut fungi dysbiosis might contribute to fungal co-infections and/or secondary fungal infection in COVID-19 patients. Aspergillus co-infection was recently isolated from the respiratory tract secretions and tracheal aspirates of COVID-19 patients.90Gut dysbiosis in COVID-19 patients was also investigated. A shotgun metagenomics analysis of 15 COVID-19 patients hospitalized in Hong Kong disclosed that their fecal microbiomes were deficient in beneficial commensals and abundant in opportunistic pathogens.93 A recent study used in-depth shotgun sequencing to investigate relative changes in the fecal virome of COVID-19 patients.27 There were increased proportions (11/19) of eukaryotic DNA viruses and decreased proportions (18/26) of prokaryotic DNA viruses  in the feces of COVID-19 patients possibly because of SARS-CoV-2 infection. The abundance of fecal eukaryotic viruses may increase to take advantage of the host immune dysfunction that may occur in response to SARS-CoV-2 infection. Analysis of the modifications in gut virome functionality revealed that in COVID-19 patients, stress, inflammation, and virulence responses were comparatively increased and included arginine repressor, hemolysin channel protein, and DNA polymerase IV expression and DNA repair.The gut virome helps regulate intestinal immune homeostasis.The foregoing studies helped elucidate the relationships between microbiota and SARS-CoV2 infection and could, therefore, disclose possible gut microbiota interventions that reduce disease severity in hospitalized COVID-19 patients.99 The symptoms of PACS may be respiratory , neuropsychiatric , gastrointestinal , dermal (hair loss), musculoskeletal  and may also include fatigue.109 The underlying reasons for the emergence of PACS are unclear. A recent study revealed that gut dysbiosis might play a vital role in PACS.110 Stool samples were collected from 68 COVID-19 patients of whom 50 (73.5%) presented with PACS at six months after the initial COVID-19 diagnosis. There was no significant correlation between fecal or respiratory viral load and PACS development. However, a six-month follow-up indicated differences in the gut microbiota between patients with PACS and those without it. The gut microbiota of patients without PACS were comparable to those of healthy controls whereas those of patients with PACS substantially differed from those of the healthy controls at six months. In addition, patients with PACS have reduced bacterial diversity and richness than the healthy individuals. In contrast, the foregoing parameters did not significantly differ between patients without PACS and healthy controls. In the PACS patients, 28 and 14 gut bacterial species had decreased and increased, respectively, compared with the healthy controls. The authors examined the associations between the gut microbiome composition and the various PACS symptoms at six months. The R package MaAsLin2 (https://github.com/biobakery/Maaslin2) revealed that different PACS symptoms were related to different gut microbiota patterns. Eighty-one bacteria were associated with various PACS classes and many of these taxa were associated with at least two persistent symptoms.Post-acute COVID-19 syndrome (PACS) is characterized by long-term complications and/or persistent symptoms following initial disease onset.Bifidobacterium, Brautia, and Bacteroidetes. Patients with PACS displayed significantly lower gut bacterial diversity and richness than those of healthy controls. Thirteen bacterial species including Blautia wexlerae and Bifidobacterium longum were negatively associated with PACS at six months. Hence, these species may have protective roles during recovery from SARS-CoV-2 infection. In contrast, Actinomyces sp S6 Spd3, Actinomyces johnsonii, and Atopobium parvulum were positively correlated with PACS. The authors also reported that certain bacterial species such as Ruminococcus gnavus, Clostridium innocuum, and Erysipelatoclostridium ramosum remained variable from admission to the 6-month follow-up and were associated with several PACS symptoms.The authors also investigated whether the gut microbiota profile at admission can influence PACS development. Analyses of stool samples at admission disclosed that bacterial clusters distinctly differed between patients with and without PACS. Compared with the PACS patients, those without PACS-COVID-19 presented with gut bacterial compositions that were enriched for 19 bacteria and characterized by Taken together, the foregoing findings suggest that gut microbiota composition upon patient admission may reflect the susceptibility of the individual to long-term COVID-19 complications. As millions of people have been infected during the ongoing COVID-19 pandemic, the discoveries of the preceding studies strongly suggest that gut microbiota modulation could facilitate timely recovery from COVID-19 and reduce the risk of acute PACS development.112 In the early stages of the pandemic, inflammatory cytokine storms were observed in certain COVID-19 patients.115 Cytokine storms are also known as inflammatory factor storms or systemic inflammatory response syndrome (SIRS).116 Excessive immunocyte activation releases large numbers of intracellular inflammatory factors including IL-6, IL-1β, TNF-α, IFN, and complement protein. Consequently, immunocytes mount storm-like suicide attacks on pathogens and infected cells, cause collateral damage to healthy cells and tissues, increase vascular permeability, and disturb circulation.117 The underlying mechanisms of the inflammatory factor storms induced by SARS-CoV-2 infection are assigned to the following three categories.119SARS-CoV-2 invades epidermal cells by binding the cell-surface receptors ACE2 and TMPRSS2, hijacks host cells, and undergoes self-replication. After large numbers of viruses are produced and released from the epithelial cells, innate lymphocytes such as macrophages and dendritic cells (DC) recognize and bind viral pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), and NOD-like receptors (NLRs). These PRRs induce the expression of proinflammatory factors, IFNs, and numerous IFN-stimulated genes (ISGs) Fig. .118,119F120Cells killed by SARS-CoV-2 infection release multiple danger-associated molecular patterns (DAMPs) that activate the RLRs and NLRs and, by extension, promote the expression of various proinflammatory factors.122 It also destroys the tight junctions (TJs) between epidermal cells . The analysis disclosed that the ACE2-B0AT1 complex exists as a heterodimer and the SARS-CoV-2 spike protein (S1) may interact with it.135 SARS-CoV-2-induced B0AT1 downregulation on the luminal surfaces of intestinal epithelial cells might contribute to microbiota dysbiosis which, in turn, promotes pathogen invasion and ultimately facilitates cytokine storms and COVID-19 exacerbation.134SARS-CoV-2 infection disrupts respiratory and gastrointestinal microbiota eubiosis by decreasing the proportions of probiotics and increasing the abundance of opportunistic pathogens. It damages the respiratory and gastrointestinal epithelial cell mucosal layers.lls Fig. .123 ThesInflammation is a protective immune response that helps clear sources of infection. However, chronic or excessive inflammation can cause autoimmune damage.136 Gut microbiota-derived metabolites are the main mediators of gut microbiota-host interactions that influence host immunity. Hence, we will discuss the potential mechanisms by which gut microbiota-derived metabolites modulate the host immune responses to SARS-CoV-2 infection.Gut microbiota metabolites are small molecules produced as intermediate or end products of gut microbial metabolism. They are derived either from the bacterial metabolism of dietary substrates or directly from the bacteria themselves.137 They are localized mainly to the spleen, lymph nodes, and liver. Nevertheless, they may also inhabit barrier tissues such as the lung, skin, and gut.138 They respond to pathogens via restrictive major histocompatibility complex (MHC)-related protein-1 (MR1)-mediated recognition of riboflavin derivatives produced by gut microbiota such as Bifidobacterium animalis, Bacteroides thetaiotaomicron, Lactobacillus casei, and Enterobacter cloacae.140 These microbially-derived signals affect all stages of MAIT cell biology including intrathymic development, peripheral expansion, and organ function.142 In tissues, MAIT cells integrate multiple signals and display effector functions associated with defense against infectious pathogens.143 A recent study showed that MAIT cells are highly involved in the host immune response against COVID-19.144 MAIT cells participate in both local and systemic immune responses in the airways during the early stages of SARS-CoV-2 infection. They are recruited by proinflammatory signals from the blood into the airways and rapidly promote an innate immune response against SARS-CoV-2 infection  constitute an evolutionarily conserved T-cell subset with innate functions resembling those of innate natural killer T cells (iNKT) cells.ion Fig. .Fig. 5Gu145 The authors used a reporter cell line harboring multiple type I IFN response elements to screen a library of 84 microbe-associated metabolites and found that DAT significantly affected IFN signaling. Mice administered DAT after influenza infection exhibited reduced mortality, lower viral gene expression, and decreased proportions of apoptotic cells in their airways. The authors analyzed changes in fecal and serum DAT content in antibiotic-treated mice and confirmed that their gut microbiota produced this compound. The researchers also reported that the human gut bacterium Clostridium orbiscindens degrades flavonoids to DAT. Gut microbiota-derived components such as lipopolysaccharides (LPS) also help protect lungs from viral infections.146 Recent evidence from Schaupp et al. suggested that microbiota-derived components are required to program dendritic cells (DCs) in steady-state so that they rapidly respond to pathogens and initiate immune responses against them.147 Another study showed that gut microbiota-driven tonic IFN signals in lung stromal cells protect the host against influenza virus infection.148 SARS-CoV-2 and influenza virus are similar in many ways. Thus, gut microbiota-derived metabolites and components might help inhibit early SARS-CoV-2 infection.149Deaminotyrosine (DAT) is a bacterial metabolite derived from flavonoids. It was recently demonstrated that DAT protects the host from influenza infection by initiating a type I interferon (IFN) signaling amplification loop.151 Siltuximab is a monoclonal antibody targeting IL-6R.152 Numerous studies showed that various microbial metabolites inhibit inflammation. Therefore, in this section, we will discuss the putative mechanisms by which these substances suppress COVID-19-related inflammation , and butyrate .153 SCFAs influence immune responses in the gut and those associated with peripheral circulation and distal body sites158 A recent study by Kim et al. found that the SCFAs produced by microbiota enhanced B cell metabolism and gene expression and supported optimal homeostatic and pathogen-specific antibody responses.159 SCFAs have these effects on the B cells in the gut and systemic tissues. Therefore, SCFAs derived from gut microbiota may promote anti-SARS-CoV-2 antibody production in B cells and inhibit COVID-19 development. Another study revealed that the gut microbiome of patients with COVID-19 presented with impaired SCFA capacity even after disease resolution.161 Thus, there may be a direct link between the severity of COVID-19 infection and persistent impairment of gut microbiota metabolism.Short-chain fatty acids (SCFAs) are produced by various bacterial groups. They include acetate , propionate  comprise a T-cell subset with significant immunosuppressive effects and the capacity to express Foxp3, CD25, and CD4. A variety of anti-inflammatory cytokines secreted from Treg cells can inhibit auto-inflammatory responses, and prevent pathological immune responses from causing tissue damage.163 Defective or absent Treg cell function may result in inflammatory disease.166 Butyrate can promote the differentiation of naive T cells into Treg cells by inhibiting histone deacetylase or increasing the transcription of Foxp3 promoter in naive T cells.157 Propionate activates GPR43 on Treg cells and enhances their proliferation.167 Other microbiota-derived metabolites and components also modulate Treg cells.SCFAs also inhibit inflammation by modulating various immunocytes. Butyrate promotes M2-like macrophage polarization and, by extension, anti-inflammatory activity by upregulating arginase 1 (ARG1) and ultimately downregulating TNF, Nos2, IL-6, and IL-12b.Bifidobacterium infantis-derived vitamin A or retinoic acid (RA) upregulates Aldh1a2 encoding retinal dehydrogenase 2 in DCs.170Aldh1a2-expressing DCs produce high levels of RA and this substance collaborates with transforming growth factor-β (TGF-β) to promote naive T cell differentiation into FOXP3+ Treg cells.171 The capsular component polysaccharide A (PSA) of the gut commensal Bacteroides fragilis can be transported to the gut lamina propria via autophagy-related protein 16-like 1 (ATG16L1) and the nucleotide-binding oligomerization domain-containing protein 2 (NOD2)-dependent autophagy pathway.174 Toll-like receptor 2 (TLR2) on FOXP3+ Treg cells recognize PSA signals which, in turn, induce FOXP3+ Treg cell proliferation, IL-10 production, and an anti-inflammatory state.177 Cell-surface β-glucan/galactan (CSGG) polysaccharide produced by Bifidobacterium bifidum promotes Foxp3+ Treg cell generation.178 Retinoic acid receptor-related orphan receptor gamma t  may induce proinflammatory T helper 17 (TH17) cell differentiation.179 Recent studies showed that certain gut commensals such as Helicobacter spp. and Clostridium ramosum can induce RORγt expression in Foxp3+ Treg cells.181 RORγt+ Foxp3+ Treg cells downregulate TH1-, TH2-, and TH17 cell-type immune responses.182Various gut commensal-derived metabolites and components such as vitamins, carbohydrates, amino acid derivatives, and glycolipids have been proved to modulate multiple host immunocyte subsets by different mechanisms. The immunomodulatory effects of gut commensals are potential targets for the design and administration of SARS-CoV-2 prophylaxes and therapies.183 Recent studies have manipulated gut microbiota to treat COVID-19 and its complications.190 Here, we review and discuss putative microbiota-based COVID-19 therapies , probiotics, and prebiotics have been used in the clinical treatment of various human diseases such as diabetes, obesity, cancers, ulcerative colitis, Crohn’s disease, and certain viral infections.ies Fig. .Fig. 7PoClostridioides difficile infection.194 Recently, a registered clinical trial  attempted to validate the efficacy of FMT as an immunomodulatory risk reducer in COVID-19 disease progression associated with escalating cytokine storms and inflammation. The control group is administered standard pharmacological treatments while the experimental group is also orally administered FMT in the form of 30–50 dose-in, double-cover, gastro-resistant, enteric-release frozen 60-g capsules.195 A main outcome measure is the incidence of adverse events in the safety pilot group up to day 30 after administration. Another outcome metric is the percentage of patients in the study and control groups requiring escalation of non-invasive oxygen therapy modalities such as increasing FiO2, administering high-flow nasal cannula oxygen therapy (HFNOT), continuous positive airway pressure (CPAP), or invasive ventilation, ventilators, and/or ICU hospitalization corresponding to grades 5–7 disease exacerbation on the COVID-19 performance status scale. This trial is still in progress. Nevertheless, considering the vital roles of gut microbiota in immune regulation, we believe that FMT is a possible therapeutic option for suppressing COVID-19-induced cytokine storms and inflammation.In FMT, feces or complex microbial communities derived from in vitro culture or purification of fecal material from a healthy donor are inoculated into the intestinal tract of a patient. FMT has demonstrated efficacy against colitis, diabetes, and recurrent 196 Treatment with probiotics and/or prebiotics is relatively safer and easier to prepare and administer than FMT. The National Health Commission of China has recommended the clinical administration of probiotics to patients with severe COVID-19 for the purposes of restoring and maintaining gut microflora balance and preventing secondary infection. Indeed, numerous clinical trials are validating the efficacy of probiotics and/or prebiotics at reducing COVID-19 duration and symptoms 197 in hospitalized COVID-19 patients. Investigators in a phase II randomized clinical trial  are assessing the efficacy of BLIS K12 and Lactobacillus brevis CD2 in the prevention of secondary bacterial pneumonia in patients with severe COVID-19. Another randomized trial  at Duke University Hospital is evaluating the efficacy of the probiotic Lactobacillus rhamnosus GG at preventing COVID-19 transmission and symptom development in exposed household contacts. None of the foregoing clinical trials has yet published the results. However, it has already been empirically demonstrated that certain probiotic stains have antiviral activity against other coronaviruses. Therefore, probiotics could potentially be used in the prevention and/or adjuvant treatment of COVID-19.Supplementation with microbiota-targeted substrates (prebiotics) such as specific dietary fibers and/or direct transfer of one or several specific beneficial microbiota (probiotics) are promising COVID-19 treatment approaches that modulate the gut microbiota.Lactococcus lactis strain was engineered to express and secrete the anti-inflammatory cytokine IL-10 to treat colitis.198 The biosafety of this strain was ensured by making it require exogenous thymidine for survival and IL-10 production.199 The cytokine storms caused by SARS-CoV-2 infection have a close relationship with COVID-19 severity and mortality. Hence, the design and application of similarly engineered strains to produce anti-inflammatory metabolites in the lungs and suppress proinflammatory storms could culminate in a promising COVID-19 treatment. While much further clinical study is required to validate the safety and efficacy of this technology. Direct supplementation of beneficial microbiota-derived metabolites such as SCFAs are also promising candidates for COVID-19 treatment.Advances in synthetic biology and gene manipulation are facilitating and realizing the design of microorganisms based on therapeutic requirements for COVID-19. We can now engineer symbiotic bacteria with desired functions, the ability to produce the required metabolites, and the capacity to target the correct locations in the host. A 205 For example, antibiotic-treated and germ-free mice had reduced antibody responses to the seasonal influenza vaccine.206 Therefore, in addition to the COVID-19 treatment, considering microbiota as a vital factor modulating immune responses to vaccination, microbiota-targeted interventions are a promising way to optimize the COVID-19 vaccine effectiveness. However, so for, relatively few studies have evaluated the effects of the microbiota on immune responses to COVID-19 vaccination and further work is required in this area.Emerging evidence from interventional studies and animal models suggests that the microbiota plays a crucial role in antibody responses to vaccination.207 The respiratory and gastrointestinal tracts are the primary habitats of human microbiota and targets for SARS-CoV-2 infection as they express ACE2 and TMPRSS2 at high levels.212 There is growing evidence that the substantial perturbation of these microbiota during COVID-19 is associated with disease severity and mortality and post-acute COVID-19 syndrome (PACS).213 Microbiota are powerful immunomodulatory factors in human health and disease.217 Hence, targeting microbiota manipulation is a promising strategy for the prevention and treatment of COVID-19 and PACS. Numerous clinical trials are evaluating the efficacy of adjuvant therapy with probiotics as well as other microbiota-based treatments. However, the outcomes of these clinical trials have not yet been published. Additional clinical data are required to validate the safety and efficacy of microbiota-based therapies for patients with COVID-19 or PACS.Symptoms associated with the initial phase of COVID-19 include dry cough, shortness of breath, vomiting, and diarrhea.218 Notably, the Omicron variant is not a single strain, but evolved into three lineages: BA.1, BA.2, and BA.3.219 BA.1 was once the most widely prevalent strain in the world; however, BA.2 is suggested to be more transmissible than the BA.1 and BA.2 is gradually replacing BA.1 in several countries, such as Denmark, Nepal, and the Philippines.220 The transmissibility of BA.3 is very limited, with very few cases, at most a few hundred cases. Certain studies proposed that the omicron variant can evade infection- and vaccination-induced antibodies and exacerbate existing public health risks.227 In contrast, other studies demonstrated comparatively lower hospitalization rates associated with the omicron variant than the wild type SARS-CoV-2.230 However, the differences among the omicron and wild type strains in terms of their relative impact on host microbiota alterations are unknown. Future investigations might help develop microbiota-based therapeutics customized for omicron variant infections.The SARS-CoV-2 omicron variant has recently and rapidly spread worldwide.231 Moreover, microbiota composition varies widely among individuals and populations.232 They also greatly differ in terms of their SARS-CoV-2 symptoms. Cases may range from asymptomatic to acute pneumonia.17 However, there is little data available on the associations among microbiota composition and coronavirus susceptibility. Thus, clarification of the relationships between SARS-CoV-2 susceptibility and microbiota composition may facilitate the design and deployment of prophylactic and therapeutic measures against the new SARS-CoV-2 strains. It is clear that microbiota are strongly implicated in host immune responses to various diseases including COVID-19. Nevertheless, it remains to be determined whether microbiota-based therapeutics influence COVID-19 outcome. This research focus should be prioritized as the COVID-19 pandemic continues to be severe in certain parts of the world.Not only various intrinsic host factors , but also extrinsic factors  have been shown to influence the composition of the microbiota.


Immunotherapy has been extensively explored in recent years with encouraging results in selected types of cancer. Such success aroused interest in the expansion of such indications, requiring a deep understanding of the complex role of the immune system in carcinogenesis. The definition of hot vs. cold tumors and the role of the tumor microenvironment enlightened the once obscure understanding of low response rates of solid tumors to immune check point inhibitors. Although the major scope found in the literature focuses on the T cell modulation, the innate immune system is also a promising oncolytic tool. The unveiling of the tumor immunosuppressive pathways, lead to the development of combined targeted therapies in an attempt to increase immune infiltration capability. Oncolytic viruses have been explored in different scenarios, in combination with various chemotherapeutic drugs and, more recently, with immune check point inhibitors. Moreover, oncolytic viruses may be engineered to express tumor specific pro-inflammatory cytokines, antibodies, and antigens to enhance immunologic response or block immunosuppressive mechanisms. Development of preclinical models capable to replicate the human immunologic response is one of the major challenges faced by these studies. A thorough understanding of immunotherapy and oncolytic viruses’ mechanics is paramount to develop reliable preclinical models with higher chances of successful clinical therapy application. Thus, in this article, we review current concepts in cancer immunotherapy including the inherent and synthetic mechanisms of immunologic enhancement utilizing oncolytic viruses, immune targeting, and available preclinical animal models, their advantages, and limitations. Immunotherapeutic targets have emerged in the past decade as a promising addition to the oncology treatment arsenal for selected types of cancers ,2. With With the advent of new immunotherapeutic targets and new developments in oncolytic virus (OV) immunotherapeutic applications, problems with preclinical validation and safety have emerged . ClassicAlong those lines, multiple animal models have been developed in an attempt to suffice the need for a reliable preclinical assessment of these drugs. Humanized mice, which comprise immune-deficient mice engrafted with human hematopoietic cells and therefore with a human immune system, are the most promising models . Each ofThe relationship between tumor tissues and the immunologic system has been increasingly explored with a new concept of immunologic modulation in the fight against cancer cells. The thought that tumors escape immune surveillance is not recent, but evolved significantly in the past decade with the development of immune checkpoint inhibitors (ICI), specifically the ones targeting the cytotoxic T lymphocyte antigen 4 (CTLA4) and programmed cell death 1 (PD1), that showed encouraging results in initial clinical trials ,10. IpilCTLA4 intrinsic function is closely linked to T lymphocyte modulation and a major responsibility for immunologic tolerance . CTLA4 iIn addition to intrinsic function, CTLA4 also modulates T lymphocyte activation extrinsic mechanisms through regulatory T cells (Tregs) modulation. Loss of extrinsic CTLA4 modulation is sufficient to induce aberrant T cell activation and autoimmunity. In this context, CTLA4 appears to modulate B7 ligand activation in antigen-presenting cells, possibly by a mechanism of trans-endocytosis . CTLA4 mThe PD1-PD-ligand 1 (PD-L1) pathway has shown to significantly improve survival in preclinical and clinical trials and is currently in clinical use for many types of cancer. PD-L1 is expressed by many types of cells including tumor cells while PD-L2 is expressed mainly by normal dendritic cells . Thus, tResearch has shown that the higher the mutation rate of a tumor, the more likely it is to produce neoepitopes, cell surface biomarkers, and, thus, to elicit immune response . The canOne of the key components in tumor microenvironment (TME) immunosuppression is the tumor-associated macrophages (TMA). TMAs have been recognized as one of the main contributors to ICI response failure in solid tumors ,34,35. TMacrophage activation is typically characterized by two polarizations: The M1, which is pro-inflammatory; and the M2, which is immunosuppressive. M1 macrophages are classically activated by interferon-gamma (IFNγ) and lipopolysaccharides and present anti-tumor activity induced by nitric oxide synthase and release of cytotoxic reactive oxygen species and pro-inflammatory cytokines ,39. WithMacrophages activated by interleukin-4 (IL-4), the M2 subtype, are called tumorigenic given their ultimate ability to express growth-promoting, pro-angiogenic, and extracellular matrix remodeling signals via vascular endothelial growth factor, interleukin-8 (IL-8), matrix metalloproteinase, transforming growth factor β and T cell suppression molecules ,43,44. C2, which both decreases NK cell function and causes downregulation of the XCR1 and CCR5 chemokine receptors on DCs [NK cells, another component of the innate immune system, contribute to oncolysis by phagocytosis and cytotoxic mechanisms and are APC-independent . NK actis on DCs .Although ICI therapy has demonstrated promising results in cancer treatment, response in solid tumors remains limited. Innate and acquired resistance to immunotherapy are known limiting factors . UnderstThe first step in immunologic response consists of the capture of tumor antigen from the tumor microenvironment by APCs such as dendritic cells . Once APSince an adequate immunologic response is dependent on the optimal function of multiple variables, any defect could generate impaired immune elimination of tumor cells. For instance, tumor cells with a low mutational burden are thought to produce fewer neoantigens and, thus, are less responsive to immunotherapy ,57. FurtThe mismatch repair (MMR) is an example of immunologic response biomarker that gained importance for pembrolizumab recommendation guidelines. MMR is the process responsible to correct for errors generated during DNA replication. MMR deficient tumors demonstrated hypermutational profile with strong neoantigen production that is highly immunogenic upon PD-L1 pathway inhibition. However, the neoantigen pattern of expression and anti-tumor immune response is unpredictable; thus, not all tumors will respond to PD-L1 pathway inhibition . This waAdditional mechanisms contributing to a decreased tumor immunogenic response include poor lymphocyte infiltration, resistant DC phenotypes, an abundance of Tregs within the tumor, which are immunosuppressive and hamper priming of naïve T cells, and recruitment of effector T cells.Based on intrinsic immunologic characteristics of the TME, a classification of tumor phenotypes based on their immune activity has been proposed . The firOVs present a natural or genetically engineered tropism for tumor cells that can be further enhanced to increase both innate and adaptive immune responses. Over the years, OVs, particularly adenovirus, have been continuously modified to increase tumor selectivity and minimize toxicity. OV mechanism of action is thought to occur through three main mechanisms: the primary lysis of tumor cells caused by intracellular viral replication, gene modification delivery, and the secondary increase in antigen-presenting molecules leading to an increased adaptive immunologic response. One of the main advantages is that given the selective nature of OVs, these mechanisms may be used systemically to reach tumor metastatic tissue that was not directly inoculated.Viruses and other pathogens naturally stimulate stronger immune responses than over-expressed self-antigens normally encountered in solid tumors. OVs have evolved through the years to express multiple cell receptors and lack intrinsic replication capabilities. OVs mechanism rely on virus-mediated cytolysis promoting the release of pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs), which are cancer cell death sensors . Tumor oWith a growing body of evidence in immunologic modulation for cancer therapy, OVs followed a similar trend by genetically adding immunologic capabilities. Talimogene laherparepvec (T-VEC) is a herpes-simplex virus encoding granulocyte-macrophage colony-stimulating factor (GM-CSF) and was the first OV-based immunotherapy to reach a phase III trial. It is currently FDA approved for the treatment of selected patients with metastatic melanoma that are not candidates for surgical resection. Following intratumoral administration, T-VEC induces cell lysis followed by the release of tumor antigen and subsequent circulation of tumor-specific effective T-cells. Thus, although the administration is local, the treatment effect reaches systemic levels. GM-CSF controls both myeloid differentiation and the function of mature presenting antigen cells. This effect has been replicated with adenovirus (Ad5-D24-GMCSF), which demonstrated complete tumor regression and tumor-specific cytotoxic T lymphocyte response . Figure Sipuleucel-T autologous cell-based immunotherapy vaccine FDA-approved for the treatment of hormone-refractory prostate cancer. Production is individualized by leukapheresis of the patient’s peripheral blood antigen-presenting cells followed by ex vivo antigen loading with prostate acid phosphatase (PAP) and GM-CSF enhancement . ApprovaThe combination of immunotherapy drugs has been attempted as an approach to increase tumor cell response. However, the combination of immunotherapy drugs carries concerns with increased adverse effects . The conWith the same concept of vaccine cancer therapy, authors recently reported on the use of cancer stem cells lysate-pulsed dendritic cell vaccine with induced tumor-specific humoral and cellular immunologic response ,73,74. CChimeric antigen receptor-modified T cell therapy (CAR-T) is an adoptive autologous T cell therapy strategy targeting cells or TME. CAR-T synthetically generates personalized effector T cells with a high affinity for tumor antigens independent of MHC . CAR-T tIn a different work by Watanabe et al., an engineered oncolytic adenovirus expressing tumor necrosis-alpha (TNFα) and interleukin-2 (IL2) (Ad5/3-E2F-D24-TNFa-IRES-IL2 or Ad-mTNFa-mIL2) was administered to humanized mice bearing pancreatic adenocarcinoma xenografts. Pancreatic adenocarcinoma has a highly immunosuppressive TME. The authors demonstrated the increased intensity of T cell infiltration with sustained tumor regression after combined meso-CAR-T cells and oncolytic virus therapy. Pancreatic adenocarcinoma was resistant to meso-CAR-T cells alone, but co-administration with Ad-mTNFa-mIL2 elicited tumor regression. Moreover, Ad-mTNFa-mIL2 demonstrated induced increased M1 polarization of macrophages and DC maturation compared to control adenovirus, again indicating that oncolytic therapy enhances innate and adaptive immunity .TMAs and OV interplay, however, is not completely clear and often contradictory in the literature. Although it has been reported that tumor inflammation inhibits viral replication through interferon release, some tumors have demonstrated a response to virus-based anti-tumor immune activation ,91. ThisNeutralizing anti-viral antibodies have been identified as one of the main obstacles to oncolytic virus tumor response. Neutralizing antibodies may originate from the previous contact with a similar community-based virus or can be generated upon contact with the inoculated virus, thus, ultimately leading to resistance to oncolytic therapy. A multitude of materials and delivery systems have been developed to increase OVs delivery efficiency and have been previously reviewed elsewhere . One recAdditionally, using the MB gene transfer system in healthy immunocompetent C57BL/6 mice or mice bearing a syngeneic TRAMP-C2 prostate tumor, we protected systemically delivered adenoviral vectors from the innate and adaptive immune system preventing the off-target distribution of the viruses .In sum, extensive research has demonstrated successful oncolytic virus encapsulation and neutralizing antibody escape by utilizing micro and nanoparticles. Recently, a new approach has been described using mesenchymal stem cells (MSC) carriers, known as trojan horses, which are inoculated ex vivo with oncolytic viruses. MSCs allow for antibody neutralization shielding, tumor penetration, and the advantage of intracellular replication  Figure 4Rodents have been historically the most common animals to star in preclinical cancer research. Over the years, the constant development of new rodent models, particularly mice, have allowed an improved understanding of human tumor biology, pathophysiology, and anticancer drug response. Choosing an appropriate preclinical model, however, is not as simplistic, and directly influences the predictive validity of the study. Typically, to advance from a murine to a human model of cancer research, mice models must lack immunity to avoid innate cancer cell rejection. Thus, with the advent of oncolytic virus cancer immunotherapeutic, the common mice models became an important limitation for preclinical validation. There was a need for a model that would mimic or more closely reproduce the human immunologic response to the studied pathology or drug. This need led to the development of mice xenografted with human immune and cancer cells.Humanized mouse models are primarily made from immunodeficient mice engrafted with human-derived cancer cells, while also bearing human-derived immune cells.The first humanized mice model described in the literature used mice bearing severe combined immunodeficiency disease (SCID) determined by a mutation in the protein kinase DNA–activated catalytic polypeptide . These mThe next generation of humanized mice included a knock-out of interleukin-2 receptor subunit gamma (Il2rg) that is responsible for the development of natural killer cells in NOD-SCID mice. Consequently, these additional IL2 mutations generated a mouse not only deficient in lymphocytes B and T, but also bearing defective NK cells, impaired macrophage function, complement activity, and DC function. From this model, the two most popular immunodeficient mouse models were created: The NOG mouse, which included a NOD mouse with mutated Il2rg leading to truncation of the intracellular signaling pathway; and the NSG mouse, which comprise a complete null Il2rg mutation .Humanized mice may be engrafted with different approaches, each delivering varying degrees of immunologic response. Here, we summarize the concepts of each approach.The simplest approach consists of the inoculation of human peripheral blood mononuclear cells to immune-deficient mice, known as the peripheral blood leukocyte-humanized immune system (PBL-HIS) model. Human reconstitution is obtained relatively fast ranging between 3–5 days, allowing for a robust activated human T cell lineage. The main disadvantage is the rapid development of graft-versus-host disease that inevitably takes place in 4–6 weeks. This timeline limits applicability to long-term studies ,107. ThuA more complex approach was subsequently developed to partially overcome the myeloid lineage limitation of PBL-HIS models by engrafting umbilical cord blood, adult bone marrow, mobilized peripheral blood cells, or fetal liver, known as hematopoietic stem cell-HIS (HSC-HIS). To avoid the early development of graft-versus-host disease, these mice are subjected to a sub-lethal gamma radiation dose before engraftment. This model allows reconstitution of functional multilineage human hematopoietic cells and longer over life . Time toThe bone marrow–liver–thymus (BLT) model is a higher complexity mouse model that uses fetal liver, fetal thymus, and autologous fetal liver hematopoietic stem cells allowing for a complete human immune system reconstitution. Mice in this model also require sublethal gamma irradiation before engraftment with delayed graft versus host disease development. Disadvantages are similar to HSC-HIS models, with higher relative costs, the complexity of human engraftment, time to reconstitution, and availability constraints of tissues . These cHumanized mouse models have significantly advanced research of viral infections throughout recent years. The study of oncolytic virus as immunotherapeutic agents presents particular challenges to preclinical model development due to specific immune cell functionality that needs to be assessed to reliably evaluate tumor response, all while accepting human tumor cell engraftment. As mentioned earlier, humanized mice bearing lymphoid tissue are the most commonly available, simplest approach, and valuable tool for OV and adaptive immunity research.OVs targeting innate immunity and the TMA have more limited preclinical model availability. To replicate the human myeloid cell maturation, the animal must bear not only human cells but also the stimulatory factors responsible for myeloid cell maturation. Mice strains have been developed for this objective by including human cytokines knock-in including human stimulating factors such as IL-3, IL-15, GM-CSF, and macrophage colony-stimulating factor . The addOV’s immune-oncology applications hold great promise, particularly in combination with ICIs to improve solid tumor response. Although ICIs demonstrate a significant tumor response in hematologic malignancies, the response is limited in solid tumors. OVs have been recently explored to target or express pro-inflammatory cytokines and molecules to enhance immune cell transfection within solid tumors with promising results.Many challenges need to be overcome to improve the clinical application of OVs therapy either as single or combined treatment. Clinical safety related to the genetically engineered virus depend on many factors involving the gene expression stability, large scale production and amplification, targeted delivery, evasion from neutralizing antibodies, appropriate preclinical safety evaluation, and tumor response assessment. Progressive development of OVs over the years demonstrated its multimodal functionality such as cell lysis, immunomodulation, and gene therapy. Oncologic OV’s applications have demonstrated over the years better results with combined therapy, which is likely secondary to the tumor’s heterogeneity. Thus, future clinical applications are more likely to involve multiple therapeutic modalities.This article summarized important concepts in the development of virus-based immune-oncology therapy development.