Herpes simplex virus (HSV) and varicella zoster virus (VZV) are alpha herpesviruses that establish life-long latent infection in neuronal ganglia after primary infection. Periodic reactivation of these viruses results in recurrent infections that can have significant impact on patients’ quality of life. HSV commonly causes oral and genital mucocutaneous infections whereas VZV is responsible for varicella/chickenpox and herpes zoster/shingles, but cancer patients are at particularly higher risk of complications including disseminated and visceral infections due to impaired cell-mediated immunity. While diagnosis of more common HSV and/or VZV infections is frequently clinically based, immunocompromised hosts may have atypical skin presentation or visceral involvement. Thus, diagnostic confirmation using virus-specific tests such as polymerase chain reaction or immunohistochemical staining is crucial in some cases. Oral acyclovir, valacyclovir and famciclovir are usually used for mild to moderate infections and intravenous acyclovir is the drug of choice for severe or disseminated infections. Foscarnet can be used when acyclovir-resistance is confirmed or suspected. Pharmaceutical prophylaxis against HSV and/or VZV should be considered in high-risk cancers patients. Currently, there is no commercially available vaccine against HSV, but VZV vaccines are available to prevent varicella and zoster.  Both Herpes Simplex Virus (HSV) and Varicella Zoster Virus (VZV) belong to the Herpesviridae family, whose members are large, enveloped viruses with a linear double-stranded DNA genome in an icosahedron capsid. Eight herpesviruses are known to cause infection in humans, including HSV-1, HSV-2, VZV, cytomegalovirus (CMV), human herpesvirus (HHV)-6, HHV-7, Epstein–Barr virus (EBV), and Kaposi sarcoma-associated herpesvirus . Based on the biologic properties of these viruses, they are sub-divided into three sub-families, the Alphaherpesvirinae , Betaherpesvirinae , and Gammaherpesvirinae  [The alpha herpesviruses are notable for their predilection to establish latent, life-long infection in dorsal root sensory ganglia, then reactivate periodically to cause asymptomatic viral shedding or clinical disease. Such characteristics of alpha herpesviruses undoubtedly shape the clinical manifestations that we observe with HSV and VZV . These viruses can cause infection of mucocutaneous tissues and visceral organs, resulting in a wide spectrum of clinical diseases, from asymptomatic shedding to fulminant sepsis or death. The course and outcome of the infection largely depend on the interplay between the virus and the host, including anatomic site(s) involved, viral type (HSV-1 vs. HSV-2) as well as age and immune status of the host . Cancer Infection of these viruses can have a substantial impact on quality of life. It can cause significant morbidities and even mortalities, particularly among those with immunocompromised conditions, including patients with malignancies. This review will discuss the epidemiology, biology, clinical manifestations, treatment, and prevention of HSV and VZV, with particular focus on cancer patients.Both HSV-1 and HSV-2 have a huge global burden. Per World Health organization (WHO), an estimated 3.7 billion people, around 66.6% of the world’s population aged 0–49 were infected with HSV-1 in 2016, while an estimated 491.5 million people equivalent to 13.2% of the world’s population aged 15 to 49 years, were infected with HSV-2 [Seroprevalence of HSV-1 and HSV-2 in general increases with age and varies among different populations (CDC) . HSV-1 iVarious studies have shown a higher prevalence of HSV infection or reactivation among cancer patients ,13,14,15Varicella or chickenpox represents the primary infection by VZV, after exposure of susceptible hosts to the virus. Chickenpox is extremely contagious and is thought to be transmitted through the respiratory route. Similar to HSV, VZV also establishes life-long latency in neuronal tissues, and its reactivation leads to zoster or shingles. Chickenpox affects children worldwide, but for countries that adopt vaccination programs against chickenpox, the epidemiology of VZV has changed dramatically . HistoriFor herpes zoster (HZ), it is estimated that about one out of every three people in the US will develop this viral infection, with roughly 1 million people affected per year. HZ can occur at all ages, although it mainly affects elderly adults with incidence increasing with advancing age . Much ofMost individuals experience only one episode of HZ; only a few percentages of patients will have more than one episode. In fact, recurrent cutaneous lesions are much more likely caused by HSV than by VZV. However, the risk of recurrent HZ is increased among immunocompromised patients . A detailed description of the biology of these alpha herpesviruses is beyond the scope of this article and is reviewed elsewhere ,8,25. BrThe HSV genome is about 150 kilo-basepair (kbp) and encodes about 84 proteins . The oveVZV genome is about 125 kpb in size, encoding ~75 proteins. The process of viral replication is thought to be similar to that of HSV. VZV is highly cell associated and direct cell to cell spread is the major mechanism of viral multiplication . HoweverThe innate immunity is the first line of defense mechanism against HSV and VZV infections. This includes natural killer cells, macrophages, dendritic cells, and Toll-like receptors. However, the adaptive immunity , plays the most important role in controlling these viral infections. Despite adequate levels of neutralizing antibodies against HSV and VZV, reactivation of viruses can still occur, indicating that cell-mediated immunity is crucial in controlling the severity and spread of primary infections and in reducing re-activations. In particular, the level of plasma HSV-specific CD8+ cytotoxic T cell precursors has been shown to be the best predictor for frequency and severity of viral reactivations ,3. HencePrimary infection with HSV starts with exposure of the virus to mucosa or abraded skin. After viral entry and replication in cells of the epidermis and dermis, the released virions can traverse the neuroepithelial junction and gain entry into nerve endings of the sensory neurons. The virus then reaches the neuronal cell bodies within the sensory ganglion by retrograde transport, where it can establish life-long latency. These infected neuronal cells can maintain normal cell function, while harboring the viral genome in a latent or repressed state with transcription of only a few viral RNAs. For HSV-1 infection, the trigeminal ganglia are the most common site of latency; for HSV-2 the sacral nerve root ganglia are most frequently involved. The mechanisms that control and maintain viral latency are unclear, but a virus can reactivate periodically without apparent damage of the infected neurons, travel back to the nerve endings, and infect cells of the mucocutaneous tissues resulting in either asymptomatic shedding, or recurrent infection manifested as clinical disease [Many clinical diseases have been associated with HSV. For both subtypes, primary infection  tends to be more severe than subsequent recurrences . While both HSV-1 and HSV-2 can cause genital herpes, recurrences tend to be more frequent with HSV-2, likely representing evolutionary adaptation of HSV-2 to the sacral ganglia milieu. Reactivation of latent HSV is exceptionally frequent in cancer patients, although virus reactivation seems to occur independently of cancer chemotherapy .A number of complications have been associated with HSV infection , from keVisceral organ infections can involve any sites, including the esophagus, liver, or lungs. When a single organ is involved, local HSV reactivation or extension from adjacent infection  may play a role. However, viremia may herald visceral infection, particularly with multiple-organ involvement or disseminated disease, which can be associated with high mortality in severely immunosuppressed patients. HSV can infect any part of the alimentary tract with the esophagus being most frequently affected. HSV esophagitis can be seen in immunocompetent individuals, but is much more common in immunosuppressed patients, including those with malignancy and those undergoing steroid or immunosuppressive therapy. The “stress” of cancer diagnosis and of treatment  frequently leads to HSV reactivation, which causes oral or perianal lesions that may mimic or exacerbate chemo-induced mucositis. For those patients with profound immunosuppression, such as those undergoing HCT, ulceration from HSV can be unremitting and disabling, causing significant impact to the patient’s quality of life.Typical symptoms include dysphagia and odynophagia; some may also experience gastrointestinal bleeding. Endoscopic examination usually demonstrates erosions or ulcers, typically involving the mid to distal esophagus. Since a number of infectious and non-infectious entities can cause ulceration of the esophagus, confirmation of HSV esophagitis requires immunohistochemical staining of biopsy specimens using specific antibodies against HSV. Histopathologic observation of multinucleated giant cells and/or inclusion bodies in the clinical specimen is inadequate to confirm the HSV diagnosis since other herpesviruses (such as CMV or VZV) can also cause esophagitis and produce similar cytopathic changes. In a recent case series of HSV esophagitis from a single center in Taiwan , 47 patiHSV pneumonitis is uncommon except in severely immunosuppressed patients, including those with hematologic malignancies and solid tumors ,33 or inIt should be noted that demonstration of HSV-1  in respiratory tract specimens does not necessarily represent HSV infection of the respiratory tract, as HSV reactivation in the oropharynx leading to contamination of the sputum or bronchoalveolar lavage (BAL) samples is relatively common. Instead, confirmation of HSV pneumonitis requires histopathological or cytological examination of the clinical specimen, such as demonstration of typical HSV cytopathic effects in BAL fluid and further confirmation of the infection with immunocytochemical staining. HSV hepatitis is a rare condition mostly reported among third trimester pregnant women and immunocompromised patients . In a reHSV-1 encephalitis is the most common cause of sporadic fatal encephalitis in the US and worldwide and is associated with significant morbidity and mortality despite antiviral therapy. Although HSV encephalitis is predominantly diagnosed in immunocompetent hosts, this entity may be under-recognized in immunocompromised patients. Its diagnosis in the latter group, such as those with cancer undergoing treatment or HCT, is often delayed due to atypical presentations; in particular, their immune-suppressed status may render the cerebrospinal fluid (CSF) results misleading. For example, in a case series of HSV encephalitis among seven cancer patients, CSF white blood cell (WBC) count was normal in four . Others Patients with HSV-1 encephalitis may present with seizures, altered mental status, and fever. For those presenting with new neurological symptoms following WBRT, the possibility of HSV-1 encephalitis must be considered to prompt early initiation of antiviral therapy.Immunocompromised individuals have a higher incidence of both chickenpox and HZ as well as their associated complications.Chickenpox is generally a benign, self-limited disease in immunocompetent children, and associated mortality is fewer than 2 per 100,000 cases, but the mortality risk increases by more than 15-fold for adults. The interval from exposure of the non-immune child to the appearance of the rash is about 14 days, with a range of 10–21 days; the incubation period may be somewhat shorter in immunocompromised children. The presenting manifestations of chickenpox are skin rash, fever, and malaise. The characteristic vesicular exanthem is often referred to as “dew drop–like” during the early stages of formation; if the vesicles do not rupture, the fluid content then becomes purulent in appearance. Skin lesions first appear on the trunk and face and rapidly spread centrifugally to involve other areas of the body. Among healthy children, crops of lesions continue to appear for about 3 days (a range of 1–7 days), with the appearance of maculopapules, vesicles, and scabs in varying stages of evolution. This phase is often prolonged in immunocompromised children, and the duration of healing the cutaneous lesions can be extended by a minimum of threefold . Skin lesions from chickenpox can be secondarily infected by bacteria, most often with Gram-positive organisms. Other than cutaneous involvement, the most frequent complication of chickenpox is of the central nervous system, which can be manifested as acute cerebellar ataxia or encephalitis. While the former is usually benign and resolves within 2 to 4 weeks, the latter can be associated with severe outcomes. Mortality is estimated at 5–20% and neurologic sequelae is observed in ~15% of survivors . VaricelHZ complications are frequent and can present with extra disease burden in cancer patients . HZ usuaWhile HZ can affect any dermatome, reactivation from the thoracic and lumbar ganglia are most common. Less common, but noteworthy sites of VZV reactivation include those of the cranial nerves (CN), which can be associated with significant complications. Reactivation from the first branch of the trigeminal nerve (CN V) can lead to HZ ophthalmicus; clinical findings may include conjunctivitis, keratitis, anterior uveitis or rarely, pan-ophthalmitis. When the second branch of CN V is affected, oral lesions of the palate may develop without any cutaneous lesions. VZV reactivation from the geniculate ganglion of CN VII can lead to Ramsay Hunt syndrome, which is described as a triad of ipsilateral facial paralysis, otalgia, and vesicles near the ear and auditory canal. Given the proximity of the infection to the vestibulocochlear nerve, some patients can also suffer from debilitating hearing loss, tinnitus, and vertigo. A number of neurologic complications have been described with HZ, including aseptic meningitis, encephalitis, transverse myelitis, granulomatous cerebral angiitis, and so on. When the anterior horn cells are involved, inflammation and necrosis may follow with resulting motor deficits. Stroke syndromes secondary to infection of cerebral arteries are also well-described  and it iHZ in immunocompromised patients can be more severe than immunocompetent hosts. In addition to more prolonged phase of cutaneous eruption, risk for cutaneous dissemination and visceral involvement  are higher. However, although patients with disseminated HZ may become severely ill, it is rarely fatal even in immunocompromised patients, especially with prompt initiation of effective antiviral therapy as discussed below.While a clinical diagnosis of HSV infection can be inferred when characteristic vesicular lesions are observed in oral or genital areas, herpetic lesions may also resemble ulceration from other causes. For cytologic or histopathic findings, HSV causes characteristic multinucleated giant cells and/or intranuclear inclusions, which can be demonstrated by cytologic staining methods such as Tzanck preparation. However, such a method lacks sensitivity, and cannot differentiate cytopathic changes with HSV from other herpesviruses such as VZV or CMV. Laboratory confirmation can be achieved by demonstration of viral antigens using direct florescent antibody (DFA) or viral DNA by PCR techniques.Prior exposure to HSV can also be demonstrated by serologic assays. Most commercially available assays measure antibodies to viral glycoproteins gG1 and gG2, which are antigenically distinct between the two subtypes of HSV, allowing differential detection of HSV-1 versus HSV-2 antibodies. Of note, IgM is not very specific for primary infection. IgM was detected in 100% of the patients with primary infections of HSV-1 and HSV-2, but also in 68% of those with nonprimary infections . AnotherChickenpox and localized cutaneous shingles are commonly diagnosed by history and a physical exam that demonstrates the presence of a diffuse vesicular rash at different stages and of a vesicular rash with dermatomal distribution, respectively. However, VZV reactivation/infection in immunocompromised hosts might have an atypical presentation and are more difficult to diagnose clinically. Detection of VZV by viral culture is not clinically useful, due to low sensitivity and long turnaround time. DFA testing of scrapings from skin lesions can provide a quick diagnosis of chickenpox or shingles, but sensitivity is limited by the quality of the specimen. In the modern era, PCR has become the test of choice to confirm VZV infection in different organ systems due to its quick turnaround time and high sensitivity. PCR assays can be used for a number of clinical specimens including skin lesions, CSF, BAL, plasma, ocular fluid, lung tissues, and so on.Serologic testing for VZV is used to establish immune status through IgG positivity; however, IgM is not useful to diagnose acute infections, as it is commonly positive in asymptomatic recurrent infections and is not specific to primary disease .All currently licensed systemic antiviral drugs for treatment of HSV and VZV work by inhibiting viral replication by means of inhibition of the DNA polymerase or the viral helicase-primase complex. A comprehensive review of antivirals with activities against the alpha herpesviruses is beyond the scope of this review and readers are referred to recent reviews on the subject ,48. AntiAcyclovir is a guanosine analogue and valacyclovir is an oral L-valyl ester prodrug of acyclovir with 3–5x better bioavailability as compared to oral acyclovir . These d2 given every 8 h was significantly superior to placebo in treating culture-proven mucocutaneous HSV infection. On average, acyclovir treated group shed HSV virus 14 days less, had lesions scabbing and pain disappearing 4 days faster, and total healing occurred 7 days quicker compared to placebo group [Early studies have demonstrated the efficacy of acyclovir in the treatment of HSV infections. In patients with underlying leukemia, lymphoma, and HCT recipients, intravenous (IV) acyclovir 250 mg/mbo group . Based obo group . HoweverFor HSV visceral infections, it has been demonstrated that patients with solid tumors and proven, probable, or possible HSV pneumonitis had lower mortality rates when treated with HSV directed antiviral therapy . SixteenThe most common mechanism of acyclovir resistance in HSV arises from the mutation of viral thymidine kinase, while the mutation of viral DNA polymerase is much less frequent . ResistaWhile chickenpox and HZ are frequently self-limiting in immunocompetent patients, acyclovir therapy can substantially reduce VZV-attributed morbidity in immunocompromised hosts . It has In cancer patients with localized dermatomal HZ, oral acyclovir at a higher dose  is an acceptable antiviral agent if patient tolerates oral therapy . HoweverAcyclovir-resistant VZV infections are more prevalent in immunocompromised hosts and the most common mechanism of resistance is deficiency in viral tyrosine kinase . HoweverPenciclovir is a guanosine analogue similar in mechanisms of action, activity, and spectrum to acyclovir. In a clinical trial that compared IV penciclovir to IV acyclovir in immunocompromised patients with severe mucocutaneous HSV infections requiring hospitalization, there were no differences in time regarding healing, cessation of viral shedding, resolution of pain, or treatment failure among groups that received either drug . PenciclFamciclovir is an oral diacetyl ester prodrug of 6-deoxy-penciclovir with high bioavailability. When comparing famciclovir 500 mg orally three times a day to acyclovir 800 mg orally five times a day (both given for 7 days) in immunocompromised patients with localized dermatomal HZ, there were no significant differences between the groups in the time to cessation of new lesion formation, healing of lesions, or resolution of acute phase pain. Famciclovir was well tolerated, with a safety profile comparable to that of acyclovir . FamciclPenciclovir and famciclovir are not recommended for treatment of acyclovir-resistant HSV isolates due to the high level of cross resistance. Foscarnet is a pyrophosphate analogue DNA polymerase inhibitor. It is active against human herpesviruses, including HSV and VZV. Due to its significant adverse effects , its use for HSV and VZV is limited to treatment of severe or disseminated infection with proven or suspected resistance to acyclovir . A dosinBrivudin is a thymidine analogue with potent antiviral activities against HSV and VZV. The inhibitory concentration of brivudin on VZV is lower than acyclovir and penciclovir  and it wCidofovir is a monophosphtate nucleotide analogue DNA polymerase inhibitor that does not require activation by viral kinases. It has broad antiviral activity against double-stranded DNA viruses, including the herpesviruses. Due to its toxicity profile, this drug is reserved for treatment of HSV resistant to acyclovir and/or foscarnet, commonly dosed at 5 mg/kg/dose weekly for 2 weeks followed by every other week or 1 mg/kg/dose three times a week, both intravenously. Topical cidofovir has been used in immunocompromised patients with multidrug resistant HSV mucocutaneous infections . Brincidofovir (CMX-001) is a lipid ester prodrug of cidofovir with significantly enhanced bioavailability as well as lower rates of nephrotoxicity and myelotoxicity as compared to cidofovir. It is currently licensed for the treatment of smallpox. It has been used in the treatment of acyclovir-resistant VZV but experience is limited . Ganciclovir is a synthetic analogue of 2′-deoxy-guanosine. It is available in both oral or IV formulations. Valganciclovir is a L-valyl ester prodrug of ganciclovir with significantly improved bioavailability. Ganciclovir is the first line antiviral treatment for CMV and also has activity against HSV and VZV. However, it is not generally used for the treatment of HSV and VZV infections due to increased toxicity compared to acyclovir.Amenamevir (ASP2151) and pritelivir (AIC316) are novel oral helicase-primase inhibitors, with a new mechanism of action distinct from those involved in inhibition of the viral DNA polymerase. Amenamevir inhibits both HSV and VZV while pritelivir is active against HSV only. None of these drugs are currently licensed in the US.Amenamevir was shown to have equivalent efficacy for the treatment of recurrent genital herpes ulcers  and cutaPritelivir is characterized by long half-life (50 to 80 h) and can be administered once weekly . In a phIn addition to antiviral therapy, good skin and wound care to prevent superimposed secondary bacterial infections are crucial for the treatment of cutaneous VZV infections. Adequate pain control is also essential for treatment of acute neuritis and PHN .For PHN, multiple analgesic approaches can be employed in the chronic management of this devastating syndrome, including but not limited to opiates, antiepileptics such as gabapentin and pregabalin, tricyclic antidepressants, and topical therapies such as lidocaine and capsaicin . In addiConcomitant steroid use with antiviral agents for the treatment of acute HZ remains controversial. In immunocompetent patients, earlier studies showed minimal improvement of quality of life and only slight benefits when adding oral steroids to acyclovir ,75, but Various types of cancers have been associated with increased risk of severe HSV and VZV reactivation/disease. The national comprehensive cancer network (NCCN) guidelines for prevention and treatment of cancer related infections  classifiSimilar to NCCN guidelines, the Infectious Diseases Society of America (IDSA) and the American Society of Clinical Oncology (ASCO) guidelines recommend antiviral prophylaxis with nucleoside analogues  for HSV-seropositive patients undergoing leukemic induction therapy or allogenic HCT . There wIn VZV-seropositive patients undergoing allogenic HCT, acyclovir prophylaxis significantly reduces risk of VZV reactivation, with hazard ratio of 0.16 compared to placebo, when given up to one year after transplantation ,79. Low Different biologics and cancer therapies also place cancer patients at heightened risk of HSV or VZV reactivation. HSV/VZV prophylaxis is recommended with the use of purine analogues , anti-CD52 monoclonal antibody , janus kinase inhibitors , anti-CCR4 agents  as well as steroids (for GVHD treatment) ,76. AntiIn adults, varicella zoster immune globulin  is approved for post-exposure prophylaxis to prevent varicella. It should be given to at-risk individuals as soon as possible after exposure to varicella or HZ and within 10 days . In a laCurrently, there are no approved vaccines for HSV. Multiple prophylactic and therapeutic vaccine trials have failed to illicit an adequate immune response against HSV-1 and HSV-2 . There aAn Oka strain-based live attenuated vaccine is approved for varicella vaccination, as a single antigen varicella vaccine  or in combination with measles, mumps, the rubella vaccine (MMRV vaccine) . VarivaxAged ≥ 13 years and by ≥3 months for patients aged 1–12 years, is recommended if there is sufficient time prior to initiating immunosuppressive therapy. It should not be administered to highly immunocompromised patients and it should be administered to eligible immunocompromised patients as the single antigen product, but not combined with MMR vaccine . Per curZostavax (Merck) is a live-attentuated zoster vaccine based on the Oka strain, with only limited data for efficacy and safety in cancer patients receiving chemotherapy. However, cases of Zostavax causing disseminated VZV infection with associated mortality have been reported among immunocompromised patients ,92,93. AIn 2017, the U.S. Food and Drug Administration (FDA) approved an adjuvanted recombinant HZ subunit vaccine  in adults 50 years of age and older. This approval was based on two concurrent randomized, placebo-controlled, multicentered controlled trials studying the efficacy of this vaccine in immunocompetent adults ≥50 years old  and adulMultiple subsequent studies have shown that Shingrix induces strong and long-lasting humoral and cellular immunity against HZ . SpecifiAnother trial recruited 606 participants ≥18 years of age with hematological malignancies who were receiving or finished immunosuppressive cancer treatments . InterveA phase 2 study by Pleyer at al. showed that bruton tyrosine kinase inhibitors  in CLL patients did not significantly affect recall immune response following vaccination with Shingrix . As a reCurrently, there is limited data about the safest timing of Shingrix vaccination after an episode of HZ disease. According to guidelines from various countries, recommendations regarding the timeframe for vaccination following an HZ are variable, ranging from after resolution of acute phase of HZ to at least 1 year . These dOther than the above mentioned licensed vaccines against VZV, there are multiple other vaccine candidates in different stages of development with at least one in a Phase 1 study .HSV and VZV are highly prevalent viruses that can cause a wide spectrum of infections in cancer patients. Immunocompromised individuals are at increased risk of such HSV and VZV infections and their complications due to their deficient immune response. Early detection and treatment initiation are essential to limit associated morbidity and decrease mortality. A number of antiviral agents are effective in treating these infections in cancer patients, while prevention through prophylaxis and VZV vaccination are crucial in reducing incidents and severity in at risk individuals.While these herpesviruses are important human pathogens, our understanding of their biology has also allowed their development into therapeutic tools. Genetically modified HSV-1 is now employed as oncolytic viral therapy to treat a variety of cancers, with examples including talimogene laherparepvec (T-VEC) for melanoma (FDA-approved) , G207 fo


Viroids are the smallest plant pathogens, consisting of a single-stranded circular RNA of less than 500 ribonucleotides in length. Despite their noncoding nature, viroids elicit disease symptoms in many economically important plant hosts, and are, thus, a class of pathogens of great interest. How these viroids establish disease within host plants, however, is not yet fully understood. Recent transcriptomic studies have revealed that viroid infection influences the expression of genes in several pathways and processes in plants, including defence responses, phytohormone signalling, cell wall modification, photosynthesis, secondary metabolism, transport, gene expression and protein modification. There is much debate about whether affected pathways signify a plant response to viroid infection, or are associated with the appearance of disease symptoms in these interactions. In this review, we consolidate the findings of viroid–host transcriptome studies to provide an overview of trends observed in the data. When considered together, changes in the gene expression of different hosts upon viroid infection reveal commonalities and differences in diverse interactions. Here, we discuss whether trends in host gene expression can be correlated to plant defence or disease development during viroid infection, and highlight avenues for future research in this field. Pospiviroidae and Avsunviroidae. These families are distinguished based on conserved sequence motifs present in viroid genomes, their mode of replication and the secondary structure they form through internal base-pairing [Viroids are small 246–434 nt), single-stranded RNAs which are responsible for diseases in many economically important crops. More than 30 species of viroids have been discovered to date, and these have been grouped into two viroid families—the -pairing . Disease-pairing . Due to 46–434 nt-pairing .Advancements in the field of genomics and transcriptomics have created new avenues for research into the molecular mechanisms of viroid disease. Microarray analyses have been used to study global changes in host gene expression induced by the potato spindle tuber viroid (PSTVd) , citrus To date, the results of viroid–host transcriptome studies have not been consolidated in a single integrative review. The most recent review of plant transcriptomic responses to viroid infection was by Owens et al. , who repPlants have evolved a complex immune response to combat diseases caused by pathogens. These responses include two lines of defence—pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI), a basal defence response, and effector-triggered immunity (ETI), a more robust immune response . Both tyPR genes in various hosts, including tomato, cucumber, hop, avocado, peach, orchid and Etrog citron [PR genes (which have varying functions in plant defence) in these interactions provides evidence for the activation of PTI in response to viroid infection.One common host response to infection by plant pathogens is the increased accumulation of pathogenesis-related (PR) proteins as part of systemic acquired resistance (SAR) in plants ,22,23. Tg citron ,27,28,29g citron ,31,32. TR genes—such as the tomato mosaic virus resistance protein, RPM1 (resistance to Pseudomonas syringae pv. maculicola 1) and RPM-like proteins, DRLs (disease resistance-like proteins), RGAs  and other nucleotide-binding site leucine-rich repeat proteins (NBS-LRRs)—upon viroid infection [R genes in response to pathogens which are unable to express effector proteins is surprising, since it is doubtful that R proteins can directly recognise the viroid RNA. We would hypothesise that the increased expression of R genes in these interactions is an effect of the activation of linked defence responses (such as the upregulation of WRKYs), rather than a strategic counterattack by infected host plants.A second plant defence response which is common to most plant–pathogen interactions is the production of resistance (R) proteins, which are usually expressed in plant hosts to detect pathogen effector proteins ,39. Sevenfection ,33,34,36FLS2), in Etrog citron [CERK1) and EF-Tu receptor (EFR) genes were upregulated when tomato plants were infected with PSTVd.Protein kinases regulate signalling pathways in all living organisms by altering the phosphorylation status of their target proteins in response to cellular signals ,41. One g citron , cucumbeg citron  and tomag citron ,29. Górag citron  also demPRRs) such as FLS2 during viroid infection is an interesting observation, and may indicate that viroid RNA is somehow recognised as a PAMP by plant hosts—an idea which has been suggested by Flores et al. [PRRs upon viroid infection is not necessarily correlated to an active function of RLKs during these interactions. Further studies are needed to investigate whether any of the upregulated receptors are able to recognise viroid dsRNA. The discovery of RLKs that bind to viroids in infected plants would provide a possible explanation for the activation of other PTI responses in these interactions.The expression of pattern recognition receptors  are a group of proteins which bind to promoter elements on the plant genome to modify the expression of host genes involved in various cellular processes, most notably those which play a role during plant–pathogen interactions ,53. SeveTaken together, transcriptome data from viroid-infected hosts reveal the upregulation of an abundance of plant defence genes across all interactions. This finding is rather curious when it is considered that viroids, unlike all other plant pathogens, do not encode proteins that could trigger PTI and/or ETI. The obvious trends in the expression of defence genes in these interactions, regardless of the viroid strain or host cultivar, led us to postulate that immune activation is most likely a result of the common mechanism of viroid pathogenesis. It has been suggested that viroids might elicit these immune responses due to their similarity with long noncoding RNAs (lncRNAs) ,55. LncROther studies have suggested that viroids elicit immune responses through direct interactions of viroids with host proteins by an unknown mechanism . HSVdPhytohormones are a group of organic molecules found in all plants which act as chemical messengers to regulate various pathways within cells. These molecules were first noted for their critical roles in plant growth and development, but they have since become the focus of many plant–pathogen interaction studies due to their role in plant defence ,60,61. DPAL), for example, was upregulated in tomato roots in late PSTVd infection (49 days post-inoculation (dpi)) [PAL was repressed in PSTVd-infected hot pepper cultivar Djulunska shipka (severe symptoms) [PAL at distinct stages of symptom development in tomato, and in pepper cultivars with different symptom severities upon PSTVd-infection, suggest that the regulation of the SA pathway may be related to the appearance of symptoms in viroid disease.Salicylic acid (SA) is a key phytohormone in plant defence against biotrophic and hemibiotrophic pathogens, due to its role in the stimulation of programmed cell death (PCD) in response to pathogen attacks ,65. SA an (dpi))  and in sn (dpi)) . In contymptoms) , and in ymptoms) . The uniSimilar to SA signalling, the jasmonic acid (JA) pathway functions in the regulation of physiological processes in plants as well as in immunity. However, its interplay with the SA pathway means that components of JA signalling often have opposing expression profiles after pathogen infection ,68,69. T(LOX) gene family encodes enzymes which function early in the JA biosynthesis pathway [LOX genes showed increased expression in hop plants infected with HSVd and HLVd [LOX expression found the gene to be downregulated [LOX. This indicated that decreased LOX expression upon viroid infection may not necessarily influence the level of JA present in the infected plant.The lipoxygenase  pathway . Four diand HLVd . In contegulated ,24,27,28egulated , howeverJAR1, which encodes an enzyme responsible for the coupling of JA to amino acids such as isoleucine, had opposing expression patterns in viroid-infected hop depending on which plant tissues were sampled. This gene was downregulated in hop leaves infected with CBCVd or HLVd, though it was upregulated in CBCVd-infected hop flowers and in HLVd-infected hop cones [JAR1 also had intriguing expression profiles in tomato upon PSTVd infection. In the susceptible cultivar Rutgers, JAR1 was upregulated in infected plants [JAR1 expression may be of importance when investigating mechanisms of tolerance and susceptibility of tomato to PSTVd. However, further analyses would be required to determine whether varied expressions of the JA biosynthesis genes coincide with an altered accumulation of endogenous JA levels in affected host plants.op cones . JAR1 ald plants ,28, whild plants . The duaEthylene (ET) is a small signalling molecule linked to JA signalling with functions in plant growth and development, and responses to stress . Unlike S-adenosyl-L-methionine synthetase (SAM1), for example, which acts early on in ET biosynthesis, was shown to be repressed in PSTVd-infected tomato at 17 dpi, but transcriptionally activated at 49 dpi [ACC) synthase (ACS) and ACC oxidase (ACO), were upregulated in tomato leaves infected with PSTVd [ACO also had contrasting expression profiles in two different cultivars of pepper infected with PSTVd [t 49 dpi ,28. Two th PSTVd , but dowth PSTVd . ACO alsth PSTVd . ET leveAux/IAA genes, for example, were revealed to be repressed in HSVd-infected sweet cherry, PSTVd-infected tomato and PLMVd-infected peach [IAA9, IAA3 and ARF8b (auxin responsive factor) was specific to the plant organs being studied [ARF8 was upregulated in leaves, but not tubers, of PSTVd-infected potato [Auxin is a key regulator of several physiological processes in plants, including growth, senescence, the formation of fruit and roots, leaf abscission, and stress responses ,74. Pathed peach ,28,36, b studied . Similard potato .It is plausible that variable expression patterns of auxin-related genes across viroid–host interactions may be linked to differences in symptoms observed for separate interactions, due to the role of auxins in physiological processes. One study suggested that the repression of auxin signalling genes may be related to the stunting symptom caused by CEVd infection of Etrog citron . Other rGibberellins (GAs) are organic molecules which have long been known for their vital functions in plant growth and development by regulating various processes such as stem elongation, seed germination, flowering and fruit growth ,76. OnlyContrasting expression patterns of GA pathway genes may indicate that changes observed here were associated with symptom development in viroid–host interactions. We hypothesise that the downregulation of the GA pathway in some viroid–host interactions is linked to the reduced plant growth that is a common symptom of Pospiviroid infection ,78. ReduPP2C), which negatively regulates ABA signalling, was downregulated in early infection (17 dpi) of tomato leaves with PSTVd [Additional phytohormone pathways that may be affected during viroid infection of plants are those related to biosynthesis and the signalling of brassinosteroids (BRs), abscisic acid (ABA) and cytokinins (CKs). The altered expression of genes involved in one or more of these phytohormone biosynthesis pathways, respectively, has been observed in viroid-infected tomato, sweet cherry, chrysanthemum and hop ,14,24,28th PSTVd . CK pathIn addition to phytohormones, secondary metabolites (SMs) can also function as cellular messengers. SMs most notably mediate the interactions of plants with their environment and are not directly involved in normal growth processes . SM prodFlavonoids are phenolic compounds with diverse functions in plants, such as UV protection, the regulation of flower pigmentation, antioxidant activity and defence against invading microbes . Genes iPhenylpropanoids are known for their roles in plant defence—mostly due to their influence on the production of flavonoids, phytoalexins and lignin . Lignin Terpenes compose a diverse group of organic compounds that are subdivided according to their chemical structures. In plants, these compounds may determine scent, attract insect pollinators, deter herbivores or play a role in defence against pathogens . SeveralIn addition to the activation of immune signalling networks, and the use of chemical messengers/compounds to defend against pathogens, plants may also respond to microbial attack by physically altering components of host cells. The plant cell wall serves as a physical barrier to protect plants from microbes, and it has been observed to be modified in plants in response to pathogen invasion ,93. BesiCellulose is a carbohydrate, composed of β-1,4-glucan, which confers structural strength to the plant cell wall . In planThe altered expression of genes encoding expansins was also noted during viroid infection ,28. ExpaPhotosynthesis is an essential process in the primary metabolism of plants, since the use of light energy to perform carbon fixation is the only way that plants can acquire the carbon they need to produce organic molecules within cells. Photosynthesis is, thus, linked to many other metabolic pathways in plants, as the carbohydrates produced during photosynthesis form the building blocks of many important cellular compounds such as amino acids, phytohormones and polymers that compose the plant cell wall ,103. It Studies analysing transcriptomes of plants infected with viroids have shown significant changes in the expression of photosynthetic pathway components in some interactions . CommonlFrom a physiological perspective, it is understandable that photosynthesis would tend to be downregulated during viroid infection, since the activation of defence responses (as discussed earlier for these interactions) can be a costly exercise for plant hosts. One possibility is, thus, that photosynthesis is downregulated during infection to conserve energy needed for plant immune responses ,102. AnoTransport proteins are involved in the movement of all chemical compounds throughout the plant—it is, therefore, reasonable to assume that genes encoding these proteins would be differentially expressed in plant–pathogen interactions, where multiple other processes are affected ,106. PlaTransporters with modified transcription after viroid infection include ATP binding cassette (ABC) transporters, amino acid transporters, sugar transporters and ion transporters. ABC transporters are proteins that have important roles in the transport of phytohormones and secondary metabolites ,108,109.The fact that modified transport activity was common in almost all transcriptome studies reviewed here illustrates that changes to transport pathways seem to be a response shared by all viroid-infected hosts. Since none of these transcriptome studies analysed gene expression in transport pathways in detail, however, it remains unclear to what extent modified transporter activity plays a role in viroid–host interactions. Future studies into the magnitude of changes observed in transport pathways might help elucidate how viroid infection affects cellular transport in host tissues.The phenotype of host plants is determined by the proteins present inside cells, of which the abundance is ultimately determined by the expression of genes that encode them. In eukaryotes, the post-transcriptional modification of mRNA transcripts, the translation of mRNA into proteins and the post-translational modification of proteins are all steps which may influence the accumulation of proteins. Additionally, the abundance of proteins is also affected by the rate at which they are degraded inside host cells at any given time. Transcriptome studies of viroid-infected hosts suggest that viroid infection may influence various steps in this process, from gene expression to protein degradation .The transcription of genes encoding TFs was shown to be affected in almost every global expression analysis of viroid-infected hosts . This isSeveral viroid–host studies noted changes in the expression of specific TF families. TF families showing altered expression levels following viroid infection most often include the bZIP family, bHLH family, MYB family, WRKY TFs, NAC TFs, C2H2 zinc fingers, ERFs and the C2C2-Dof family ,90,112. Eukaryotic pre-mRNAs undergo extensive modification before translation. One of the key processes in the post-transcriptional modification of RNA is RNA splicing, where noncoding regions of the RNA (introns) are removed, and coding regions (exons) are spliced together to form a mature RNA transcript. The process of alternative RNA splicing, where different combinations of exons are maintained in the unique versions of mature mRNA, can produce a number of different mRNA transcripts, and, therefore, different proteins.One study on the transcriptome of PSTVd-infected tomato revealed that, in addition to the altered transcription of host genes, the mRNA of certain protein-coding genes was alternatively spliced upon viroid infection . AuthorsOnce mature mRNA has been produced, it is ready to be translated into a polypeptide chain. Through the process of RNA silencing, the abundance of mature mRNA transcripts may also be regulated. Briefly, this process involves the use of double-stranded RNA (dsRNA) or hairpin RNA (hpRNA) as a substrate for the enzyme Dicer or Dicer-like (DCL), which then cleaves the dsRNA or hpRNA into smaller RNA duplexes (sRNAs) 20–24 nucleotides long. These sRNAs may be classified as either small-interfering RNAs (siRNAs) or micro RNAs (miRNAs) depending on their size, function and interacting proteins. Once cleaved, sRNAs interact with Argonaute (AGO) proteins to form the RNA-induced silencing complex (RISC), which uses the sRNA to guide the complex to mRNA with a complementary sequence. AGO enzymes then cleave the targeted mRNA, effectively preventing its translation downstream ,123,124.DCL and AGO genes in HSVd-infected cucumber, PSTVd-infected tomato and CEVd-infected Etrog citron [Expression analyses in plants infected by viroids have shown marked changes in the expression of enzymes that are needed for RNA silencing. Authors noted the altered expression of g citron ,12,29. Ag citron ,126.The effect of viroid infection on RNA silencing components remains unclear, as obvious trends are not easily observed in the available transcriptomic data. It is plausible that the altered expression of genes related to RNA silencing is linked to the mechanisms by which viroids cause disease . AlteAnalyses of affected pathways in transcriptomic studies showed that genes related to the process of translation were differentially expressed upon the viroid infection of sweet cherry and tomato ,29,36. SThe majority of transcriptomic studies failed to note the modified expression of protein synthesis components as a key finding; however, when examined together, the combined transcriptomic data revealed that the manipulation of translation seemed to be common across various viroid–host combinations. To date, viroid interference with protein synthesis has not been specifically studied in any infected host, but altered levels of translation might mean that protein accumulation does not correlate to the level of gene expression observed in transcriptomic studies. Future investigations in this area should aim to support observations of differential gene expression with evidence of altered protein accumulation in affected host cells.The abundance and activity of proteins may be affected by various post-translational processes. The differential expression of genes related to protein modification, protein phosphorylation and/or protein degradation has been observed in several hosts infected with viroids . This suEarly studies of viroid-infected hosts revealed the differential phosphorylation of a tomato protein upon PSTVd infection ,128, andThe molecular mechanisms of viroid pathogenesis have been the subject of scientific investigations for over 20 years, yet much remains unknown. Several recent studies have analysed host gene expression in response to viroid infection in the hopes that this would further our understanding of these interactions. When the results of viroid–host transcriptomic studies were taken together, several cellular components and signalling networks were observed to be commonly influenced by viroid infection .The most distinctive trends in gene expression were observed for plant defence pathways, which were upregulated across all viroid–host interactions. Hormone signalling pathways appeared to be affected in all viroid-infected hosts , though Currently, the transcriptomic response of tomato to PSTVd is the only interaction studied in depth, with all variables taken into consideration. Only one or two transcriptomic studies have been carried out for each of the other viroid–host interactions reviewed here , while hAvsunviroidae [trans-acting siRNAs, which would trigger signalling cascades in host cells [Host gene expression is extensively modified during viroid infection, but it remains unclear how this expression is triggered by what Flores et al.  termed “viroidae . It is cst cells ,130. Anost cells  states tNicotiana benthamiana [One theory that has very recently been proposed is that viroids, which have always been considered noncoding entities, may have the capacity to encode peptides after all . This hathamiana . Despitethamiana ,44, whicWhile transcriptomic studies are incredibly valuable in elucidating the host response to viroids, gene expression data alone are not enough to draw solid conclusions regarding the effects of viroid infection. The host phenotype is influenced by the abundance of specific proteins, but the transcription of a particular gene might not necessarily correlate with the accumulation or activity of the encoded protein. Conclusions drawn from transcriptome data should, thus, be supported by evidence of altered protein functioning. Milanović et al.  used RT-


Modern sequencing technologies have generated low-cost microbiome survey datasets, across sample sites, conditions, and treatments, on an unprecedented scale and throughput. These datasets often come with a phylogenetic tree that provides a unique opportunity to examine how shared evolutionary history affects the different patterns in host-associated microbial communities.In this paper, we describe an R package, phyloMDA, for phylogeny-aware microbiome data analysis. It includes the Dirichlet-tree multinomial model for multivariate abundance data, tree-guided empirical Bayes estimation of microbial compositions, and tree-based multiscale regression methods with relative abundances as predictors.phyloMDA is a versatile and user-friendly tool to analyze microbiome data while incorporating the phylogenetic information and addressing some of the challenges posed by the data.The online version contains supplementary material available at 10.1186/s12859-022-04744-5. Advances in high-throughput sequencing technologies are allowing large-scale profiling of microbial communities. After quality control and data preprocessing, sequencing reads are organized into tables or matrices, in which the rows represent samples and the columns are counts of clustered sequences that represent community members . In many microbial survey studies, there is also a phylogenetic tree that depicts the evolutionary relationships among microbes based on their genetic closeness, and a metadata matrix that contains information about the samples (such as body mass index or disease status).Data from microbiome studies have proven useful for understanding the important role of microbes in human health and disease. However, analyzing and interpreting these data is challenging due to high dimensionality, uneven sequencing depth, data sparsity, and compositionality . Apart fphyloMDA takes as input a count matrix, a metadata matrix, and a phylogenetic tree. It consists of three modules: multivariate modeling of microbial counts, extraction of relative abundances from counts, and regression with relative abundances as predictors Fig. . A user Module I assumes that the multivariate count data are distributed according to Dirichlet-tree multinomial (DTM) , 6. LoosModule II of the package applies an empirical Bayes strategy to estimate the relative abundances underlying raw count data , 8. By sExtracted relative abundances, known as compositions, are used as input into downstream analyses such as diversity estimation, differential abundance testing, and compositionally aware data analysis.Linear log-contrast models are popular for regressing a univariate response on a compositional predictor . 10] in in10] inAssuming that phylogenetically close taxa have similar associations with a host phenotype,  introducModule III implements these two procedures.For illustration, we apply phyloMDA to the COMBO dataset  which, aThe (zero-inflated) DTM distribution can be used to model multivariate abundance data. The results of DTM fitting and regression can be found in Additional file Microbiome data are often normalized prior to downstream analysis. By assuming a multinomial distribution for microbial counts and a Dirichlet-tree prior for the proportions, we transform raw counts into relative abundances by using the estimated posterior mean. The results of diversity estimation and differential abundance testing on extracted relative abundances can be found in Additional file Since the compositions carry only relative information, subcompositions are fundamental objects of investigation in compositional data analysis. We consider the regression of BMI on the estimated composition and apply TASSO to select subcompositions at subtree levels. The results are shown in Fig. We have presented a new and simple-to-use tool, phyloMDA, that offers three modules for analyzing microbiome data while simultaneously incorporating the phylogenetic information and mitigating the challenges posed by the data, ranging from the modeling of multivariate abundance data to estimation of microbial compositions to regression of a phenotype onto relative abundances. Note that the phyloseq package has been developed for microbiome data analysis , and thaAdditional file 1 A user manual for the phyloMDA package


Growing evidence suggests a wide spectrum of potential cardiovascular complications following cancer therapies, leading to an urgent need for better risk-stratifying and disease screening in patients undergoing oncological treatment. As many cancer patients undergo frequent surveillance through imaging as well as other diagnostic testing, there is a wealth of information that can be utilized to assess one's risk for cardiovascular complications of cancer therapies. Over the past decade, there have been remarkable advances in applying artificial intelligence (AI) to analyze cardiovascular data obtained from electrocardiograms, echocardiograms, computed tomography, and cardiac magnetic resonance imaging to detect early signs or future risk of cardiovascular diseases. Studies have shown AI-guided cardiovascular image analysis can accurately, reliably and inexpensively identify and quantify cardiovascular risk, leading to better detection of at-risk or disease features, which may open preventive and therapeutic opportunities in cardio-oncology. In this perspective, we discuss the potential for the use of AI in analyzing cardiovascular data to identify cancer patients at risk for cardiovascular complications early in treatment which would allow for rapid intervention to prevent adverse cardiovascular outcomes. Cardio-oncology is a new frontier in cardiology that aims to improve the prevention, detection, and management of cardiovascular complications caused by cancer therapies of various mechanisms . As compRecent advances in applying artificial intelligence (AI) to routinely obtained cardiovascular data have demonstrated the ability of these sophisticated algorithms to both risk-stratify patients from simple sets of data as well as drastically improve the detection for a variety of different conditions . These pCardiac dysfunction has been associated with a wide spectrum of cancer therapies including but not limited to anthracyclines, HER2 targeted therapies, select tyrosine kinase inhibitors (TKIs), radiation therapy, and more recently immunotherapies especially in association with myocarditis . HoweverLeft ventricular ejection fraction (LVEF) changes have been primarily used to diagnose cardiotoxicity of cancer therapies. The American Society of Echocardiography and the European Association of Cardiovascular Imaging guidelines recommend averaging five consecutive beats to calculate LVEF  has been proposed as a more sensitive means for early detection of cardiac dysfunction prior to a detectable drop in LVEF. A consistent measurement of left ventricular GLS could be instrumental in guiding chemotherapeutic regimen. However, the study by Farsalinos and colleagues noted significant variability among various ultrasound system vendors when measuring GLS , troponin I, and blood urea nitrogen (BUN) , 14. ThrSimilarly, AI can identify less obvious information contained in EKG images to detect conditions like anemia which may further assist in identifying risk cohorts . EKGs arFurthermore, AI in EKG may help identify early signs of myocardial dysfunction caused by cancer therapies. Attia et al. reported that AI-guided analysis of EKG accurately and reliably identified asymptomatic LV dysfunction defined as ejection fraction ≤ 35% with sensitivity and specificity of 86.3 and 85.7% respectively . In a seJust as EKGs are useful in identifying those at risk for LV dysfunction, paired with AI analysis, they can have the potential to provide low cost and non-invasive screening for atrial fibrillation (AF) as shown by Attia and colleagues . AF is oIn addition to identifying those who might be at risk for AF, deep learning analysis of EKGs can be used to assess patients at risk for drug-induced QT prolongation . QT prolPreexisting cardiovascular diseases (CVD) and risk factors have been associated with an increased likelihood of subsequent cardiotoxicity following cancer therapies . TherefoCoronary artery disease (CAD) and cancer are both diseases found more frequently in the elderly. Presence of significant CAD increases the risk of treatment-associated complications, and the evaluations and treatment of these CAD, such as stress testing and angiograms frequently create delays for definitive therapy. A rapid evaluation of a patient's CAD risk at the time of cancer diagnosis has the potential to streamline the process and deliver timely care. One commonality among oncological patients is routinely performed cross-sectional imaging such as CT for cancer staging. Staging CT scans are traditionally not felt to be sufficient to evaluate one's risk of CAD as they are not cardiac-gated. The application of AI to non-gated imaging has recently been shown to be able to generate accurate CAC score predictions , 29. ThiIn another study, Poplin and colleagues used deep-learning models to analyze retinal fundus images and predict cardiovascular risk factors that were not previously thought to be quantifiable or manifested in retinal images, like age, gender, blood pressure, and ethnicity . CurrentFinally, Attia and colleagues have shown that AI-guided analysis of EKGs could identify patients who had normal LVEF and were at a four-fold risk of developing left ventricular dysfunction over the next 5 years and before apparent left ventricular dysfunction could manifest symptomatically . FurtherAlthough the prospect of utilizing AI and deep learning to predict patients' risks of developing cardiotoxicity from cancer treatments is exciting and promising, there are practical hurdles and methodological limitations that currently prevent these techniques to reach their full potential. Prospective validation of the utility of incorporating the AI-mediated clinical decision-making tool into routine clinical practice is needed prior to the implementation of these promising avenues into cardio-oncology practice. For this, our institutions are collaboratively working toward building a prospective, well-labeled database with predefined variables to employ available AI-based tools in analyzing cardiovascular data to predict future cardiovascular outcomes. However, there remain challenges due to the heterogeneity of treatment regimen, diverse patient populations, differences in mechanism of toxicities, and relatively small cohorts. Such challenges may preclude effective development and validations of these AI tools, and must be overcome in order for these powerful tools to reach their full potential. International, multi-center collaborations to build well-curated patient data registry, such as the recent initiative by International CardioOncology Society, may help overcome these hurdles to identify meaningful signals among these heterogeneous populations . FinallyThe use of AI in detecting vulnerable populations for cardiovascular complications due to cancer treatment is extremely important and timely in the field of cardio-oncology and has the potential to greatly improve patient care. Leveraging recent advances in AI to analyze patient images and data standardizes the procedure, generating more consistent results that don't vary from physician to physician and reduce human error. The use of AI would not only improve the accuracy of measurements such as LVEF, but it would also identify noble imaging features correlated with cardiotoxicity. With better identification of at-risk populations or patients with early signs of cardiotoxicity, clinical care can be optimized for each patient to implement cardioprotective strategies early on in treatment to achieve better cardiac and oncologic outcomes.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.HC, DO, PC, and J-WR conceived and outlined the manuscript. FJ and TB contributed to design the study and provided critical input on the manuscript. HC, PC, and J-WR wrote the manuscript with input from all authors. All authors contributed to the article and approved the submitted version.This work is supported by of National Institutes Health grants K08 HL148540 (J-WR), K08 HL153798 (PC), K23 HL145017 (TB), K99 5K99HL157421, and American Heart Association Career Development Awards (J-WR and PC).Author TB has received speaker and consultant fees from Biotronik, Medtronic, and PaceMate unrelated to this work. Author DO has received consultant fees from Ultromics, Echo.IQ, Anthem, and Alexion. Author FJ has received consultant fees from Janssen unrelated to this work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Total hip replacement (THR) is one of the most common elective orthopaedic operations. However, evidence suggests that despite postoperative pain improvements, aspects of longer-term physical performance, such as walking ability, do not reach the levels expected when compared to the general population. Walking is best assessed by using gait analysis. This review aims to explain the concept of gait analysis, its use to evaluate THR outcomes, and its proposed future importance when evaluating new technologies proposed to improve functional recovery in individuals undergoing THR surgery. Furthermore, this review discusses the advantages and challenges of gait analysis in THR patients and provides recommendations for future work. Total hip replacement (THR) is one of the most common elective orthopaedic operations, and it is predicted to increase in prevalence as the population ages. The National Joint Registry (NJR)  reportedThis review aims to explain the concepts of gait analysis and the current state of evidence in THR so that healthcare professionals not working in biomechanics on a day-to-day basis  can increase their knowledge. This will help them to understand the potential future importance of gait analysis when evaluating new technologies and approaches that attempt to improve functional recovery after surgery. Such approaches may include enhanced rehabilitation pathways and also new surgical technologies such as computer navigation, robotic-assisted surgery, patient-specific instruments (PSI), and new surgical approaches such as the direct anterior approach that propose a better functional recovery. Navigated THR surgery is an image-guided approach, usually incorporating a 3-dimensional preoperative planning simulator and intraoperative surgical navigator. Coupling optimised preoperative planning with accurate surgical navigation, this type of surgery assists the surgeon in properly orienting the component, minimising the risk of impingement and dislocations . RoboticOverall, all these new surgical technologies have a similar aim: to ensure a more accurate implant position. The implant insertion is suggested as a fundamental factor in the long-term outcome of a successful THR surgery . It is eGait analysis assesses individuals with conditions affecting their ability to walk . WalkingIn contrast, expensive laboratories with many pieces of equipment (gait labs with motion-capture cameras) are commonly used in university research settings or specialised clinical facilities. It is worth noting that the platform on which gait is assessed can be the most significant difference between these gait laboratories. Conventional gait analysis has historically utilised force plates and is performed overground, whereas more advanced gait systems now offer instrumented treadmills (with an integrated force plate) with the capability of self-selected walking speed. Instrumented treadmills are increasingly used in gait research and clinical environments, despite acknowledged limitations such as cost, requiring a designated facility, data variability, interpretation and time . These t®, Science Citation Index and Medline®. Search strategy terms were  AND  AND (robotic surgery OR navigation OR PSI). Only three articles [A computer-based search was completed in May 2022 using the mySearch Database (Bournemouth University). Articles published in the English language were reviewed. This included the Cochrane Database of Systematic Reviews library, CINAHL Completearticles ,17,18 weAll three studies reported a better precision in the placement of the implants with the robot’s assistance and presurgical planning. In two studies ,17, outcIn addition to these papers examining the effect of new THR technologies on gait, a recent review  concludeThis review aimed to explain gait analysis concepts and the current state of evidence in hip replacement to an audience with no or limited biomechanics knowledge and position its proposed future importance when evaluating new technologies to improve functional outcome measures. Gait analysis can provide necessary knowledge pre- and postop on the level of recovery of individuals post-THR surgery ,24. WhetStudies on surgical outcomes of robotic and navigation-assisted surgery provide relevant evidence to support the optimal cup positioning and higher accuracy in implant insertion in contrast to conventional THR surgical method ,16,17. HFurthermore, kinetic data could detect aberrant force transmission across a joint which may be associated with implant wear . VariablAs discussed, gait analysis can quantify movement patterns pre- and postoperatively. Still, a certain methodological quality should be considered when conducting a gait analysis. Several of our recommendations and clinical directions are as follows. First, we recommend that gait analysis is undertaken before and after surgery in order to understand individual gait changes. The first three months post-THR surgery is when a patient will experience accelerated improvement in their functional recovery, including gait . Still, This review concludes that gait analysis can be used to quantify movement patterns for both pre- and post-THR surgery and help better evaluate functional recovery in new surgical techniques such as robotic-assisted THR surgery. Currently, limited evidence suggests that despite better accuracy in implant positioning, there is no evidence of better spatial–temporal or kinematic gait data in a patient undergoing a robotic hip replacement in contrast to the conventional surgical method. Additionally, although gait was improved after surgery in each of these new surgical technologies, small differences in kinematic and spatiotemporal parameters were still reported at 6 months postoperatively between the THR patients and healthy individuals.


Drug resistance is a common barrier to continued effective treatment in cancer. In non-small-cell lung cancer (NSCLC), tyrosine kinase inhibitors that target the epidermal growth factor receptor (EGFR-TKIs) exhibit good efficacy in cancer treatment until acquired resistance occurs. It has been observed that drug resistance is accompanied by numerous molecular-level changes, including significant shifts in cellular metabolism. The purpose of this study was to critically and systematically review the published literature with respect to how metabolism differs in drug-resistant compared to drug-sensitive NSCLC. Understanding the differences between resistant and sensitive cells is vital and has the potential to allow interventions that enable the re-sensitisation of resistant cells to treatment, and consequently reinitiate the therapeutic effect of EGFR-TKIs. The main literature search was performed using relevant keywords in PubMed and Ovid (Medline) and reviewed using the Covidence platform. Of the 1331 potentially relevant literature records retrieved, 27 studies were subsequently selected for comprehensive analysis. Collectively, the literature revealed that NSCLC cell lines resistant to EGFR-TKI treatment possess characteristic metabolic and lipidomic phenotypic signatures that differentiate them from sensitive lines. Further exploration of these reported differences suggests that drug-resistant cell lines are differentially reliant on cellular energy sources and that modulation of relative energy production pathways may lead to the reversal of drug resistance. Lung cancer is the second most diagnosed cancer world-wide , with NSTargeted therapies that include EGFR-TKIs are currently an effective standard of care treatment, with the first-generation TKIs-erlotinib and gefitinib-giving patients up to one year of progression-free survival (PFS)  before aThe dysregulation of cellular metabolism has been acknowledged as a hallmark of cancer for over 90 years . Driven A multi-omics approach including genomics, proteomics and metabolomics allows for a wider comprehension of disease by combining information from diverse biological levels . While c1H-NMR) spectroscopy [In its broadest sense, metabolomics  is the study of measuring the levels of metabolites in biological samples such as cells, tissues and biofluids , allowintroscopy . LC-MS itroscopy .Lipidomics is the study of all the non-polar metabolites within a sample to obtain the lipidomic phenotype or lipid profile. Cancer cells heavily rely on de novo lipogenesis for energy production and membrane biosynthesis to maintain the rapid proliferation of cells ,20,21. IUntargeted-omics studies provide a means to generate metabolomic or lipidomic profiles that are representative of the small molecule composition of samples obtained from biological systems with minimal a priori selection of analytes, but often with a substantial requirement for annotation despite providing an efficient route to the generation of new hypothesis. By contrast, targeted-omics studies (often incorporating isotope labelling to measure metabolite flux) can be used to measure a specifically predetermined selection of small molecule metabolites, and are often a direct route to interrogating pathways of interest . MultivaThe aim of this systematic review was to give insight into the current landscape on investigating TKI resistance in NSCLC, with a focus on the metabolic adaptations of TKI-resistant cell populations.The review was conducted and written in accordance with the 2020 Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines . A literFrom the 27 studies selected for comprehensive analysis by review of the full text and all supplementary materials, three main themes were identified: (i) metabolome rewiring, (ii) reversal of drug resistance by metabolic intervention and (iii) alterations in dysregulated lipid metabolism. These are discussed in turn below. The gold standard for measuring the metabolic phenotype of cells is metabolomics; in this context to compare and contrast the metabolite profiles of EFGR-mutant treatment sensitive and treatment resistant NSCLC cells. Our review of the literature highlighted key multiple metabolic pathways altered during the onset of drug resistance.1H NMR-based metabolomic approach, Li et al., 2016, identified 36 assigned metabolites that differentiated erlotinib-resistant and sensitive cell lines [Using a ll lines . Overallll lines .Using capillary electrophoresis-time of flight MS (CE-TOF-MS), Serizawa et al., 2014, measured the metabolic profile of erlotinib-resistant and sensitive cell lines and determined 18 metabolites that significantly contributed to distinguishing sensitive from resistant cell lines . These iThiagarajan et al., 2016, used transcriptomics and metabolomics, which determined that transforming growth factor beta (TGF-ß) contributed to adaptive drug-escape, a phenomenon where cells are able to acquire new methods of resistance, along with altered metabolic bioenergetics, regulated by mitochondrial function . Mass spIn addition to drug resistance, drug tolerance can also occur . Drug toThe Warburg effect, as stated, describes a switch in energy metabolism where cancer cells adapt to gaining their energy supply from aerobic glycolysis, as opposed to oxidative phosphorylation ,8,9. FigDe Rosa et al., 2015, investigated the effects of EGFR targeting TKIs on glucose metabolism in NSCLC, reversing the Warburg effect . They foAs a preference for oxidative phosphorylation over glycolysis is a key energy metabolism hallmark of resistant cells, targeting the pathway itself would be one way to revert cells to favouring glycolysis . In a paThe metabolomic studies in To look at the dysregulation of energy metabolism in TKI-resistant cells, with the intention to re-sensitise cells to treatment and reinstate therapeutic effect, targeting proteins implicated in metabolism was an approach used by several authors ,33,34,355′ adenosine monophosphate-activated protein kinase (AMPK) is a sensory kinase activated by intracellular levels of ATP and AMP . Its dowPyruvate dehydrogenase PDHK1) is a protein complex that when activated can inhibit glycolytic activity [ is a proTargeting glutamine metabolism, a key nutrient for cancer cells, can also be a potential strategy to overcome drug resistance in NSCLC . A compoA further approach to reinstate sensitivity to TKIs and downregulate resistant cells’ requirement for glycolysis is by targeting alternative signalling pathways.Ye et al., 2017, targeted AKT expression in resistant cells in combination with inhibiting glucose metabolism . AKT andCellular metabolism is known to change when cancer cells are under hypoxic conditions, due to the lack of oxygen to the cells. This can result in drug resistance for those drugs which require oxygen to be cytotoxic, or of interest in this review, by the alteration of metabolism . In hypoTransporters play key roles in determining cellular energy fate. An upregulation of a key transporter involved in glycolysis for example, such as GLUT1, would result in more glucose being transported into the cell , and if Bach et al., 2018, investigated bone morphogenetic proteins (BMPs), which belong to the transforming growth factor ß (TGF-ß) family and control many processes including cell differentiation and tumour growth . ActivatmTORC2 was found to play a role in metabolic reprogramming in erlotinib-resistant cells, as these cells had more metabolic flexibility when compared to sensitive cells, measured via spare respiratory capacity (SRC). SRC is a measure of the extra mitochondrial availability, i.e., the ability for increased ATP production via oxidative phosphorylation, and is an indicator of metabolic reprogramming . KnockdoLipid metabolism is tightly linked to glycolysis and oxidative phosphorylation, see Chen et al., 2021, also determined a link between SREBP1 and EGFRm response to osimertinib, which found that the mature form of SREBP1 (mSREBP1) was degraded in the presence of osimertinib, suppressing lipogenesis. However, once these cells acquired resistance to osimertinib, the ability to suppress lipogenesis was lost, and mSREBP1 levels began to increase once more [Xu et al., 2021, used lipidomics to show that gefitinib treatment altered the ratio of saturated and unsaturated phospholipids in resistant and sensitive cell lines, highlighting that prolonged SREBP1 activation is key for de novo lipogenesis, and resistant cells do in fact rely upon this mechanism . AnotherCholesterol is an essential cell membrane lipid and plays an integral role in maintaining cell function and integrity . CholestSCD1 is a further key enzyme in lipogenesis, responsible for synthesizing saturated fatty acids (SFAs) into monounsaturated fatty acids (MUFAs) . These cJung et al., 2015, set out to investigate the phospholipid composition of extracellular vesicles (EVs) that tumour cells secrete . Using mThe investigation into cellular metabolism dysregulation in EGFR-TKI resistant cells compared to those cells that are still sensitive to drug treatment, can elucidate several mechanisms within key pathways that may be targeted to prevent or reverse resistance.The Warburg effect is a well characterised hallmark of cancer and alludes to the switch seen in cancer cells in which they favour aerobic glycolysis over oxidative phosphorylation, even in oxygenated conditions . HoweverThis systematic review has highlighted the importance of investigating metabolomic and lipidomic remodelling, and the pathways that are altered allowing resistant cells to dysregulate cellular metabolism. In the papers summarised here, the studies use both metabolomics and lipidomics to investigate the phenotype of resistant cells, to understand the changes observed from gene expression to metabolite abundance.By elucidating the metabolome and lipidome changes found within resistant cells and comparing these to sensitive cells, specific metabolic pathways can be targeted alongside EGFR-TKI treatment to allow continued therapeutic benefit.


Osteoarthritis (OA) remains a prevalent disease affecting more than 20% of the global population, resulting in morbidity and lower quality of life for patients. The study of OA pathophysiology remains predominantly in animal models due to the complexities of mimicking the physiological environment surrounding the joint tissue. Recent development in microfluidic organ-on-chip (OoC) systems have demonstrated various techniques to mimic and modulate tissue physiological environments. Adaptations of these techniques have demonstrated success in capturing a joint tissue’s tissue physiology for studying the mechanism of OA. Adapting these techniques and strategies can help create human-specific in vitro models that recapitulate the cellular processes involved in OA. This review aims to comprehensively summarise various demonstrations of microfluidic platforms in mimicking joint microenvironments for future platform design iterations. Osteoarthritis (OA) remains one of the most prevalent chronic musculoskeletal disorders affecting the mobility of the synovial joints, with an estimated global prevalence of 23% among middle- to old-aged individuals . The disVarious OA models, including small and large animal models, ex vivo tissue explants, in vitro two-dimensional (2D) models, and 3D cell culture models, have been developed to facilitate the mechanistic understanding of the disease and identify suitable treatments . DespiteMicroscale tissue culture devices have evolved with the development of microfabrication technologies, which enables tissue culture engineers to match physiological tissue environments . These tOA is now considered a systemic disease involving the suffering of the whole joint, including cartilage, subchondral bone plate, and synovium . Animal Post-traumatic OA is correlated with trauma, the most widely studied subtype of OA. In terms of application, this phenotype of OA is mainly studied using invasive animal models to evaluate potential medicine’s therapeutic effect and prognosis. The advantage of invasive animal models over other OA models is that it progresses rapidly, so the research cycle is shorter, more likely to cause severe lesions, highly reproducible, and less costly. Mice models commonly used to study post-traumatic OA included the DMM and MSX by performing meniscectomy on the knee meniscus. Longo et al. provided a comprehensive review of post-traumatic OA animal models . The claAnimal models possess five unique advantages compared with existing common in vitro tissue culture models . First, Overall, animal models used in OA can simultaneously help decipher cell–cell interaction and tissue-tissue interaction , cell anIn vitro OA models are an excellent substitute for animal models because they can satisfy the criteria to study the progression of OA in vitro . AdditioThree-dimensional culture is more comparable to the OA phenotype than 2D culture as their microenvironment allows more extensive cell-to-cell and cell-to-ECM interactions. For chondrocytes, a previous study showed increased chondrogenic markers  in 3D cultures compared to 2D models ,43. To mBeyond mimicking the 3D microenvironment, there is a need to capture the mechanical stress of cartilage and bone during joint movements in the daily activities of OA patients. Simulated joint movement through periodical mechanical loading in tissue constructs is observed to slow the progression of OA. For example, Pötter N. et al. showed physical force exposure on chondrocytes exhibited higher chondrogenic marker expression, such as ACAN, COMP, and COL2, contributing to cartilage matrix maintenance .Biochemical signalling molecules (growth factors and cytokines) also play an essential role in dictating the progression of OA. Inflammatory factors such as IL-6 and IL-1ß  are knowMicrofluidic organ-on-chip (OoC) is an emerging technology comprising bioengineered human tissues housed within microfluidic devices. OoCs leverage microfabrication technologies to achieve spatio-temporal control over various cell microenvironmental factors at the cellular length scale (between 10–100 microns)  and, theWith growing evidence of the importance of 3D cultures in providing physiologically relevant tissue response , most ofFor cartilage models, hydrogels were predominantly in OoCs ,72,73,74Liquid-phase hydrogels are typically loaded through a straight channel with microstructures to guide the hydrogel flow. For example, Paggi et al. reported a microfluidic device with channels consisting of lined micropillar arrays, partitioning chondrocytes laden agarose within the microchannel, which were then perfused with culture media . This paA different technique for capturing chondrocyte 3D tissue configuration is densely packing the cells into micro-compartments. Dense-packed chondrocyte pellets, commonly thought to mimic mesenchymal condensation , showed Incorporating scaffolds to create 3D tissue models for chondrocytes and bone are also a common strategy ,80,81. TSince different aspects of tissue microenvironment can be achieved across the common culture modalities , demonst−2 [2) [With growing evidence of the role of physical activities as a potential treatment of OA, modelling mechanical signalling in microfluidic bone and tissue culture either induced by interstitial fluid motion or through forces subjected during joint loading could provide a suitable animal alternative to screen treatment efficacies for OA. With almost all microfluidic devices holding a volume of less than 500 μL, microfluidic cultures almost always relied on perfusion to facilitate the mass transport of nutrients during cell cultures, which generate fluid-induced shear stress. The shear stress can be controlled by manipulating channel geometries  and flow rate guided by the Hagen–Poiseuille equation , with a −2 , corresp−2 . The per−2 , inflamm−2 , ECM syn−2  as well −2 . Bao et −2 [2) . It was Apart from liquid shear stress, cartilage and bone tissues are also subjected to consistent mechanical stress during joint motion. Various attempts have been reported to generate mechanical stress within microfluidic devices suitable for cartilage tissue models. For example, Paggi et al. used a pneumatic channel network alongside the cartilage chambers . The choOne of the hallmarks of OA disease pathology is the inflammation response to the cartilage and bone tissue in both acute and post-traumatic injury. During acute joint damage, the regulation of cytokines, such as IL-1, IL-6, and IL-10, within the joint synovium affects the regulation of cartilage metabolism . In the Liquid mixing within microfluidic devices remains diffusion dominant due to the inherently low Re number (<100). It is common for microfluidic channels to contain flow dividers separating fluid streams to generate a chemical gradient, subjecting cells to a range of chemical concentrations depending on their position. Several microfluidic devices use this mechanism to generate polarised tissue cultures  and perfTo date, several demonstrations have integrated these strategies to tune growth factor exposure to the cartilage culture for OA models, which was covered extensively in the previous review by Banh et al. . For exaIn addition to controlling soluble growth factors, drugs, and cytokines, there is a need to tune the dissolved oxygen environment for OA. The mimicry of the hypoxia region in microfluidic joint models is particularly important since healthy articular chondrocytes rely on hypoxia to retain their metabolism and matrix synthesis ,55. EvidStrategies for generating hypoxia locally for OoC tissue cultures remain limited , with thVarious OoC co-culture models were established to capture the cross-talk between chondrocytes, osteoblasts, osteoclasts, and macrophages as a potential OA model . With caSynoviocytes have been found to play a key role in regulating OA inflammation cascade via proinflammatory factors such as nitric oxide and prostaglandin E . InflammTherefore, the pairing of synoviocytes and chondrocytes in OoCs would enable mechanistic investigations into inflammation regulation within the synovium. For example, Rothbauer et al. co-cultured synoviocyte and chondrocyte connected by a perfusion channel  to studyOoC co-cultures of bone/cartilage with mesenchymal stem cells (MSCs) can be a potential application to identify stem cell-based therapies. MSC’s immunomodulatory role through EVs has been suggested for application as a treatment option for OA ,120. At Neovascularisation during OA involves multiple cell types , which can potentially be studied in OoC co-cultures. The neovascularisation process is known to be assisted through VEGF signalling by the osteoblast ,122. In There is minimal control of the biochemical environment in many of these models due to the circulation of shared culture media within the culture device .The growing interest in translating OoCs for large-scale manufacturing processes highlights the use of PDMS as OoC fabrication material is no longer ideal for the next generation of OoC, compared to their plastic counterparts such as PEGDA and polycarbonates, heralded as materials compatible with high throughput fabrication and rapid prototyping. This shift in paradigm in fabrication material has significant consequences for microfluidic OoC. In particular, the guided flow of hydrogels requires hydrophobic surfaces (from the PDMS) to generate surface tension between the loaded hydrogels and neighbouring channels. In addition to the reliance on hydrophobic PDMS, many OoCs still depend on flexible PDMS to actuate and induce mechanical stress across tissue cultures. Therefore, additional considerations, such as altering device fabrication to incorporate a flexible membrane, should be considered for joint OoCs in an approach similar to that demonstrated by Ingber’s group . AlternaFabrication materials aside, the current OoC setup is still limited to 2 cell type co-cultures. A multi-compartment OoC  can be uThe complexities of OA pathophysiology mean the modelling of joint tissue can be achieved either by creating organo-typic tissue constructs by carefully selecting cell models or by controlling the tissue microenvironment. Both strategies have yielded varying degrees of success for their potential application in OA disease modelling and drug testing applications, along with their challenges for implementation. At this point, it is still difficult to model all the microenvironments in a single OoC. A systematic study of joint OoC tissue behaviour in relation to animal models could help identify the importance of microenvironment aspects in relation to different stages of OA.With OoC positioned as the next in vitro model, it is essential for tissue engineers to consider fabrication materials and processes that are suitable for large-scale manufacturing. It is equally important for tissue engineers to consider the user’s perspective in manufacturing, handling, and operating these OoCs to improve their compatibility with common lab equipment, such as incubators and microscopes. Approaches to reduce the need to assemble tubing into OoCs are widely popular for their portability ,130,131.The combination of flexible material with hydrogel cultures of joint OoC is potentially useful for future investigation of different stages of OA. For instance, by controlling the PDMS stiffness and pneumatic pressure, different degrees of mechanical stress can be applied to OoC tissue culture to induce injury, mimicking the onset of OA due to mechanical overload . SeparatIn conclusion, OoCs today provided an effective tool to mimic and control the tissue microenvironment and allow systematic examination of the OA mechanism, effectively independent of animal models. OoCs offer flexibility to mimic multiple tissue environments and can provide physiologically relevant tissue responses compared to standard tissue culture practices. Adapting new fabrication techniques and biomaterials is expected to add value for OoCs in the drug discovery and disease modelling process.