BK virus nephropathy in kidney transplantation is widely recognized as animportant cause of graft dysfunction and loss. In the case of transplants oforgans other than kidney, BK virus nephropathy in native kidneys has beenrecognized as a cause of chronic kidney disease, which is related withimmunosuppression; however, the diagnosis is usually late because the renaldysfunction is attributed to other causes, such as toxicity by anticalcineurinicdrugs, interstitial nephritis due to medications, hemodynamic changes, diabetes,hypertension, etc. We report a case of BK virus nephropathy in a patient whounderwent heart transplantation due to peripartum cardiomyopathy. The kidneybiopsy reported active chronic tubulointerstitial nephritis associated with latestage polyomavirus nephritis and the blood viral load for BK virus was positive(logarithm 4.5). The immunosuppressive treatment was reduced, and after twoyears of follow-up, the patient had stable renal function with a serumcreatinine of 2.5 mg/dL (GFR of 23.4 mL/min/1.73m2). We recommend that the BKvirus be considered as a cause of renal dysfunction in heart transplantrecipients, with the aim of detecting its replication in time to reduceimmunosuppressive therapy before irreversible compromise of renal function maymanifest. Reactivation of the virus can occur in immunosuppressedpatients, particularly in patients receiving immunosuppressive therapies,,At present, the exact route of transmission is not known, but it is believed that thevirus is usually acquired via the air in childhood,In recent years, cases of BK virus nephropathy have been reported in native kidneysof patients who have undergone bone marrow transplantation, as well as intransplantation of other non-renal solid organs, such as heart, liver, and lung2 during the first year post-transplant. Management wasexclusively performed by the cardiac transplant group and routine monitoring ofpolyomavirus with viral load BK or urine cytology was not done. In 2016, shepresented an elevation of serum creatinine up to 1.9 mg/dL, with a GFR of 32.6mL/min/1.73m2. At that time, tacrolimus trough level was 7.2 ng/mLand everolimus, 5.2 ng/mL. Toxicity by anticalcineurinics was suspected; therefore,tacrolimus was reduced to 4 mg daily and creatinine value returned near to thebaseline value ; no kidneybiopsy was performed. In March 2017, creatinine raised to 2.69 mg/dL, with a TFG of21.4 mL/min/1.73m2, for which she was hospitalized. The patient statedshe did not present any symptom. At physical examination, she was observed in goodgeneral condition, heart rate of 80 beats per minute, blood pressure of 130/90 mmHg,respiratory rate of 15 per minute, afebrile. Additional studies were conducted:ultrasound of the renal tract showed normal renal size, but increased echogenicity;urinary microscopy and culture analyses were negative, with no hematuria, pyuria orcasts; echocardiogram with adequate cardiac function; serological tests for HIV,syphilis, hepatitis virus B and C were negative; tacrolimus trough level of 5.2ng/mL, and everolimus of 5.98 ng/mL  of 102mL/min/1.73m98 ng/mL . Managem2.The kidney biopsy revealed active chronic tubulointerstitial nephritis, associatedwith late stage polyomavirus nephritis . PCR for14, and it can lead to terminal chronic kidneydisease of the native kidneys,Polyomavirus nephropathy is a severe opportunistic infection that occurs in kidneytransplant patients. In rare cases, it also affects the native kidneys of transplantrecipients of other organs8,16. Vigil D et al. reviewed theliterature on heart transplants with BK virus infection. Eleven patients werereported, nine males, 81% of cases associated with rejection, of which 72% hadterminal chronic kidney disease, with a mortality of 27%8. In the casereported here, the patient was a heart transplant recipient, with a previous episodeof acute rejection, so she was strongly immunosuppressed. In addition, she presenteda progressive increase in creatinine, initially attributed to toxicity byanticalcineurinics, but due to the poor response to the initial therapy, a renalbiopsy was performed, and viral load was requested for BK virus, which was positive.These findings allowed the diagnosis of BK virus nephropathy, and due to the latediagnosis, the patient developed stage 4 chronic kidney disease,,In the context of cardiac transplantation, BK virus infection of the urinary systemhas been reported mainly during rejection episodes, associated with an increase inimmunosuppressant drugs. In some studies, BK viruria of up to 19% has been reportedin heart transplant recipients, and viremia up to 5%, but BK virus nephropathy isunusual in this population. It has been suggested that additional damage to thenative kidneys is required for the development of BK viralnephropathy12 but thisstudy was not performed on the patient because initially this diagnosis was notconsidered. Electron microscopy in urine samples has a sensitivity and specificityof 100% but is not available in many centersDetection of tubular or urothelial cells with inclusions of BK virus cells in urineis a useful tool for the diagnosis of this infection in the urinary system. Thesecells are known as Decoy cells for their similarity with tumor cells in Pap smears.Decoy cells have a sensitivity of 25% and a specificity of 84%,-Kidney biopsy is considered the "gold" diagnostic standard. It is usually indicatedwhen viremia is greater than 10,000 copies/mL, with or without creatinine elevation,and with kidney dysfunction without recognizable causein vitro , butthey have not yet shown significant results in clinical studies,,Currently, there is no standard therapy for BK virus nephropathyIn summary, BK virus nephropathy in transplant patients other than kidney transplantrecipients is a silent entity that can lead to chronic kidney disease with increasedmorbidity and mortality. We therefore propose to consider the BK virus infection asa cause of renal dysfunction in heart transplant recipients, with the aim ofdetecting its replication in time to reduce immunosuppressive therapy beforeirreversible impairment of renal function may manifest.


SLC26A9 contributes to epithelial Cl− secretion, thus preventing mucus obstruction under inflammatory conditions. Additionally, SLC26A9 was identified as a CF gene modifier, and its polymorphisms were shown to correlate with the response to drugs modulating CFTR, the defective protein in CF. Here, we aimed to investigate the relationship between SLC26A9 and CFTR, and its role in CF pathogenesis. Our data show that SLC26A9 expression contributes to enhanced CFTR expression and function. While knocking-down SLC26A9 in human bronchial cells leads to lower wt- and F508del-CFTR expression, function, and response to CFTR correctors, the opposite occurs upon its overexpression, highlighting SLC26A9 relevance for CF. Accordingly, F508del-CFTR rescue by the most efficient correctors available is further enhanced by increasing SLC26A9 expression. Interestingly, SLC26A9 overexpression does not increase the PM expression of non-F508del CFTR traffic mutants, namely those unresponsive to corrector drugs. Altogether, our data indicate that SLC26A9 stabilizes CFTR at the ER level and that the efficacy of CFTR modulator drugs may be further enhanced by increasing its expression.SLC26A9, a constitutively active Cl These− transporter [A popular alternative therapeutic strategy has been the activation of other (non-CFTR) channels/transporters, as it would apply to any individual with CF, irrespective of the CFTR mutations, i.e., a “mutation-agnostic” approach. An example of one such potential therapeutic target is Solute carrier family 26 member 9 (SLC26A9), a member of the SLC26 family of multifunctional anion transporters . SLC26A9nsporter  and is pnsporter ,16, thusnsporter ,18,19. Snsporter ,21,22, ansporter . Furthernsporter . Moreovensporter , and pro− transporter, its localization was found to be mainly cytoplasmic or at the tight junctions (TJ) level [The exact direct role that SLC26A9 plays in CF and in other respiratory disorders is, however, yet to be elucidated. Although it is described as a ClJ) level . It was J) level ,28. NameJ) level .− secretion in CF but also to increase the effect of the already available CFTR modulator therapies.Altogether, these data not only highlight the impact of SLC26A9 expression in lung disease and response to CFTR modulator drugs but also motivated our research on the interaction between SLC26A9 and CFTR. Thus, our main goal here was to better understand the interplay between these two proteins, and how SLC26A9 might be used to develop novel CF therapies to be applied on their own or in combination with those currently available to further enhance their effects. Our data first confirmed that SLC26A9 and CFTR co-localize in human native lung tissue, including in CF epithelia where SLC26A9 expression is reduced. We then demonstrated that in native non-CF lung epithelia SLC26A9 localizes at the TJ level as well as in the sub-apical compartment. In human bronchial (CFBE) cells, we showed that SLC26A9 expression levels strongly affect CFTR expression and function. Particularly, F508del-CFTR rescue by HEMT is influenced by SLC26A9 expression. Interestingly, SLC26A9 overexpression did not significantly alter the PM expression of other CFTR traffic mutants, namely those that are not rescued by HEMT. This suggests a role for SLC26A9 in stabilizing CFTR at the ER, thus increasing the pool available to traffic along the secretory pathway and reach the PM. Taken together, these data support a role for SLC26A9 as a clinically relevant disease modifier and promising therapeutic target not only to circumvent deficient ClTo address the interaction of SLC26A9 with CFTR in human airways, we first investigated their subcellular localization in native human airway tissue. To this end, we stained healthy control and F508del/F508del bronchi sections for SLC26A9, CFTR and tight junctions (TJ) marker zonula occludens-1 (ZO-1). In control tissue, SLC26A9 appears to co-localize with CFTR , arrows,Thus, these data support the co-localization of SLC26A9 with CFTR and ZO-1 in human native airway tissue: either apically in non-CF tissue, or as a diffuse sub-apical localization in CF (F508del/F508del) tissue.p = 0.01 for day 7 wt vs. F508del, Based on this co-localization of CFTR and SLC26A9 with TJ protein ZO-1, we assessed how CFTR and SLC26A9 expression levels vary over the course of differentiation by Western Blot (WB) in primary human nasal epithelial (pHNE) cells derived from an individual with CF homozygous for the F508del mutation and from a non-CF control. In non-CF pHNE cells we observed that CFTR expression increased during differentiation, while SLC26A9 expression is constant over the course of differentiation, it still showed a trend towards higher expression levels after 7 and 14 days in ALI conditions . HoweverNext, we aimed to confirm how reducing SLC26A9 expression influences CFTR expression, trafficking, function, and the response to HEMT, i.e., the combination of correctors tezacaftor (VX-661) with elexacaftor (VX-445). We stably transduced CFBE cells expressing wt- or F508del-CFTR see  with a sWe then evaluated the effect of SLC26A9 KD on CFTR function by Ussing chamber. Despite that, SLC26A9 KD was modest in CFBE wt-CFTR cells ~31%, C, cAMP-aTo confirm the effects of SLC26A9 KD on CFTR expression and function in a system expressing endogenous CFTR, we then stably transduced the 16HBE cell lines expressing either wt- or F508del-CFTR with the same shRNAs targeting either SLC26A9 or Luciferase (control) and assessed the effect of all the clinically available CFTR correctors VX-809, VX-661 and VX-445. As expected, total wt- and F508del-CFTR protein expression were significantly reduced, and the rescue of F508del-CFTR PM expression (band C) by VX-445 alone, or in combination with VX-661, was also diminished . SLC26A9These data suggest that SLC26A9 KD  promoter system.Given the negative impact of SLC26A9 KD on wt-CFTR expression and function, and importantly, on F508del-CFTR rescue by the clinically available modulators, our next step was to examine the effect of increasing SLC26A9 levels on F508del-CFTR mediated ClAfter adding doxycycline (Dox) to induce the SLC26A9 construct expression in F508del-CFTR CFBE cells, we could detect SLC26A9 A,C by WBSimilar data were obtained in 16HBE cells overexpressing SLC26A9 thus further validating these results and demonstrating that SLC26A9 enhances total and PM expression of endogenous wt-CFTR  and rescInterestingly, lowering SLC26A9 expression (by KD through shRNA transfection) resulted in a delay in cell polarization of CFBE wt-CFTR cells, as seen by the lower values of transepithelial resistance (TEER) on day 3, eventually reaching the levels of the control cells on day 5 . No diffSince F508del is classified as a class II mutation, i.e., has impaired trafficking , we deciIn the present work we demonstrated the synergistic effect of SLC26A9 expression levels on CFTR expression and function, and most importantly, on the rescue of F508del-CFTR by the HEMT currently available in the clinic (VX-661 + VX-445). Consistent with previous data , our datAlthough thus far, the co-localization of SLC26A9 and CFTR has been assessed mostly through their heterologous expression either in HEK293T or CFBE41o-cells transfected with tagged SLC26A9 constructs ,26,28, rImportantly, it was established that SLC26A9 and CFTR behave differently in polarized vs. non-polarized cells , namely Also consistent with these data on diffuse SLC26A9 expression at the compromised TJ structures in CF , our res− transport. F508del-CFTR rescue by the clinically available HEMT was also inhibited in both cell models by SLC26A9 KD. On the other hand, SLC26A9 overexpression increased F508del-CFTR rescue by HEMT (expression and function), confirming that it is possible to further improve the current best treatment for people with CF carrying F508del. Enhancing SLC26A9 expression could therefore increase Cl− secretion in two ways: (i) by its own function as a Cl− transporter , and (ii) by its synergistic rescuing effect with the HEMT VX-661 + VX-445.To further investigate the relationship between SLC26A9 and CFTR we used two different cell models: CFBE, which stably overexpress CFTR; and 16HBE, with endogenous CFTR expression. We took advantage of molecular biology tools to decrease (shRNA) or increase (cDNA) SLC26A9 expression levels in these cells. Consistently in both systems, SLC26A9 KD led to a reduction in wt- and F508del-CFTR protein expression and in CFTR-mediated Cl+/K+ ATPase (data not shown).Given ours and previous data showing the effect of SLC26A9 expression in PM-localized CFTR  and theImportantly, despite that some class II mutations tested here (namely G85E and N1303K) showed a significant—albeit modest—correction by Trikafta in studies in primary nasal epithelial cultures from CF patients , we ackn− secretion, and/or by enhancing the effect of the already clinically available HEMT.In summary, our data revealed that SLC26A9 expression is crucial for enhanced CFTR PM expression and function, while also having a potential effect on TJ structures. Moreover, increasing SLC26A9 levels is a potential therapeutic option for CF, by providing an additional source of Clv/v) CO2.Immortalized Human Bronchial Epithelial cells (16HBE41o-) endogenously expressing wt-CFTR (16HBE wt-CFTR) or genetically modified to express F508del-CFTR (16HBE F508del-CFTR), and Human Embryonic Kidney (HEK) 293T were grown in Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS). Cystic Fibrosis Bronchial Epithelial cells (CFBE) expressing wt, F508del, G85E, R560S or N1303K-CFTR were grown in MEM supplemented with 10% FBS and 2.5 µg/mL puromycin. These cells were generated by lentiviral transduction of CFBE 41o-  cells. Primary human nasal epithelial (pHNE) cells were obtained from nasal brushing of healthy individuals or people with CF with an F508del/F508del genotype. Nasal brushings were obtained from Hospital Santa Maria and Hospital Dona Estefânia  after receiving patients’ written consent and approval by the hospital Ethics Committee. After isolation, cells were expanded and differentiated according to protocols established by Jeffrey Beekman’s lab  . Brieflyv/v, Electron Microscopy Sciences, 15710) and then dehydrated and slowly frozen. After lung cleaning and fixation, the pieces of secondary/tertiary bronchi were kept for 12 h at a time in phosphate buffers with increasing sucrose  content  and then incubated in a final solution with 15% (w/v) sucrose and 7.5% (w/v) gelatine  for 1 h at 37 °C. Dry ice-chilled isopentane  was then used to slowly freeze the tissues, which were kept at −80 °C until sectioning. Tissue sections were cryocut using a Leica CM1850 UV cryostat. Cryosections 15–30 μm thick were generated on Superfrost® Plus slides , and used right away for immunohistochemistry, or stored at −20 °C until further use.Explanted CF lungs (F508del/F508del) and healthy control tissues were collected in the Paediatrics Department of Motol University Hospital  under approval of applied regulations and the hospital’s Ethics Committee and shipped over 24 h to Lisboa. Informed consent was obtained from all subjects. The explanted lungs were dissected in order to identify the secondary and tertiary bronchi by removing the connective tissue using a scalpel and surgical scissors. The bronchi were then washed with cold PBS several times to remove the mucus and cut into small fragments followed by overnight fixation with electron microscopy grade PFA  0.2% (v/v), after which tissue autofluorescence was quenched with NaBH4 . A blocking step with BSA 1% (w/v) was performed before incubating overnight at 4 °C with primary antibodies. The following primary antibodies were used: rabbit polyclonal anti-SLC26A9 , mouse monoclonal anti-CFTR , and mouse monoclonal anti-ZO-1 . The following day a mix of the secondary antibodies  and nuclear dye  was applied for 2 h at room temperature (RT). Filter sections were mounted in a mix of N-propylgallate  and Glycerol for microscopy . The tissues stained were secondary/tertiary bronchi and were as similar as possible for comparison. Areas of extensive shedding/remodeling in CF tissue were avoided in the analysis, and areas of intact epithelia preferred. Imaging was performed with a Leica TCS SP8 confocal microscope coupled to a Hamamatsu Flash4 sCMOS camera, using an HC Plan Apo 20×/0.75 objective. Software used for acquisition was Leica’s LAS x, and image processing was performed on ImageJ FIJI [Lung tissue sections on SuperfrostgeJ FIJI . FIJI wa® shRNA from Sigma, Germany) or the cDNA of GFP-tagged SLC26A9 in a Tet-On 3G inducible expression system. HEK cells were seeded at a density of 5 × 105 cells per well of a poly-lysine coated 6-well plate and incubated for 24 h at 37 °C, 5% CO2 (v/v). Then, cells were transfected with 5 µg of DNA per well—2.38 µg of packaging plasmid pCMV-dR8.74psPAX2, 0.24 µg of enveloping plasmid VSV-G/pMD2.G, and 2.38 µg of the plasmid containing the shRNA/cDNA. The cells were then incubated for 18 h at 37 °C, 5% CO2. The medium was replaced to remove the transfection reagent and the cells were incubated for 30 h at 37 °C, 5% CO2. The media containing the lentiviral particles were harvested and the packaging cells were discarded. The harvested viral particles were immediately used to transduce CFBE or 16HBE cells seeded the day before on 6-well plates. Cells were infected with 2 mL of lentivirus-containing medium, plus 4 µg/mL of Polybrene ) infection enhancer. The plates were centrifuged at 200× g for 1 h at 25 °C and then incubated for 24 h at 37 °C, 5% CO2. The medium was then changed to the original cell medium supplemented with selection antibiotics  to eliminate the non-transduced cells. In the case of SLC26A9 overexpressing cells, 1 μg/mL doxycycline (Dox) was added 48 h before experiments to induce its expression.HEK 293T cells were used to produce lentiviral particles containing shRNAs targeting either SLC26A9 or Luciferase (shLuc) as a control (MISSIONSemi-quantitative reverse-transcriptase (RT)-PCR was performed to quantify SLC26A9 gene knockdown. Total RNA was isolated using the NZY Total RNA Isolation kit . Total RNA (1 μg/20 μL reaction) was reverse-transcribed using random primers and NZY M-MuLV Reverse Transcriptase . Each RT-PCR reaction contained sense and antisense primers for SLC26A9 (0.5 µM) or for GAPDH (0.5 µM), 0.5 μL cDNA and NZYTaq II DNA polymerase . After 2 min at 95 °C cDNA was amplified during 30 cycles for 30 s at 95 °C, 30 s at 56 °C and 1 min at 72 °C. PCR products were visualized by loading on RedSafe Nucleic Acid Staining Solution (Intron Biotechnology) containing agarose gels and analyzed using Image Lab (BioRad).w/v) polyacrylamide gels and transferred into polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 1 or 5% (w/v) non-fat milk powder (NFM) in Tris buffer saline with Tween 20 (TBS-T) or in PBS-T for 1 h at RT and incubated overnight at 4 °C with primary antibodies  diluted 1:250 in 1% NFM/PBS-T or mouse CFTR 596 antibody (Cystic Fibrosis Foundation (CFF), 1:3000 in 5% NFM/TBS-T). Membranes were also probed with 1:3000 mouse anti-α-tubulin in 5% NFM/PBS-T  as loading control. The membranes were incubated with HRP-conjugated goat anti-rabbit/anti-mouse IgG (diluted 1:3000 in 1% or 5% NFM/TBS-T or NFM/PBS-T) for 2 h at RT. Chemiluminescent detection was performed using the Clarity™ Western ECL substrate  and the Chemidoc™ XRS system (BioRad). The quantification of band intensity was performed using the Image Lab software (BioRad) and normalized to the loading control as appropriate.Protein extracts from CFBE or 16HBE cells were separated on 8.5%  and 1.12 cm2 area. Transepithelial electrical resistance (TEER) of the monolayers was measured with a chopstick electrode  and electrophysiological analyses were carried out in monolayers with resistance values around or above 600 Ω × cm2. Transepithelial resistance (Rte) was determined by applying 1 s current pulses of 0.5 µA (5 s-period). For Ussing chamber measurements, Snapwell inserts were mounted in the chamber device and continuously perfused with Ringer containing (mM): NaCl 145, K2HPO4 1.6, MgCl2 1, KH2PO4 0.4, Ca-Gluconate 1.3, Glucose 5, pH 7.4. Low Cl− ringer (NaCl 30 mM) was added to the luminal side to create an electrochemical driving force. Experiments were performed in the presence of Amiloride (20 μM), an inhibitor of the Epithelial Sodium (Na+) Channel (ENaC). CFTR was activated by the adenylyl cyclase activator Forskolin (2 µM) and Genistein (25 µM) added to the luminal side and inhibited by the specific inhibitor CFTR inhibitor 172 (30 µM). Values for the transepithelial voltage (Vte) were referenced to the luminal epithelial surface. Equivalent short-circuit currents (Ieq-sc) were calculated according to Ohm’s law from Vte and Rte (Ieq-sc = Vte/Rte), with appropriate correction for fluid resistance.CFBE or 16HBE cells were seeded at approximately 2.5 × 10t-test (for paired or unpaired samples as appropriate) or ANOVA were used for statistical analysis. A value of p ≤ 0.05 was accepted as statistically significant. The number of experiments performed (n) is indicated in figure legends.Data are reported as means ± SEM. Student’s


General personal information, physical condition, TCMBC, biochemical, and SNPs were collected for eligible subjects. The body constitution questionnaire (BCQ) was used to evaluate the relationships between TCMBC tendency, biochemical values, and obesity-related SNPs.  The adults with body mass index (BMI) more than 27 kg/mn = 33, 66.0%); however, TCMBC in combination is not uncommon (30 subjects with more than two TCMBC in combination). For biochemical profiles, leptin was higher among patients with yang deficiency constitution (YangDC)  and YinDC . The leptin level was highest among YangDC subjects. Higher leptin was found among subjects with three-combined TCMBC than balanced TCMBC subjects who were not inclined to any of three TCMBC. For obesity-related SNPs, the adrenergic receptor beta-3 (ADRB3) gene tended to be high expression among YangDC  and uncoupling protein 1 (UCP1) tended to be high expression among phlegm-stasis constitution (PSC) .  Obesity patients tended to have a yin deficiency constitution (YinDC) ( The relationships between TCMBC, leptin, and SNPs present alternative viewpoints about TCMBC and could be used as a guide to treat obese patients. Obesity is a complex, chronic medical condition with a significant negative impact on human health . Since tThere are many reasons for obesity, which can be divided into congenital and acquired. In addition to the acquired causes such as dietary habits, physical activities, and living habits, the heredity of the genetic constitution is also valued by scholars . The herFor traditional Chinese medicine (TCM) doctors, the constitution is one of the most important clinical references to treating diseases. The constitution is broadly defined as the fundamental components that constitute a human being and includes the total expression of physiological, psychological, and pathological traits that characterize a person's health . It is aThe present study aimed to investigate the association between TCMBC and obesity-related polymorphisms in obese patients. Our results could help to identify abnormal constitutions with a high risk for obesity and help clinicians focus on preventive strategies more effectively on these patients.2; (2) age ≥ 20 years; (3) no recognition disabilities; and (4) willing to sign informed consent and tolerance to blood sampling and questionnaire filling. The exclusion criteria were the following: (1) pregnant women or planning to conceive; (2) a history of endocrine disorders other than diabetes mellitus; (3) history of psychiatric disease and not compliance to blood sampling or questionnaire filling; (4) impaired activity of daily living; and (5) record of other ongoing clinical trials.We prospectively conducted this study from January 2018 to December 2019 at the Department of Traditional Chinese Medicine of Chang Gung Memorial Hospital . The age, sex, BMI, waist-hip ratio, waist-height ratio, lifestyles, obesity-related genotyping and biochemical profiles, and comorbidities were collected, and TCMBC was measured. This study was approved by the Institutional Review Board (IRB) of Chang Gung Memorial Foundation (IRB no.: 201701723B0). All the study participants gave informed consent and signed a therapeutic partnership agreement. The inclusion criteria were the following: (1) BMI ≥ 27 kg/mα of each constitution's subscale ranged from 0.55 to 0.88, with the intraclass correlation coefficients exceeding 0.7 [Professor Yi-Chang Su kindly approved the use of the body constitution questionnaire (BCQ), Ph.D., School of Chinese Medicine, Chinese Medical University , and BCQ has been widely used for TCM constitution assessments , 18. Allding 0.7 , 18, 21.The following variables were recorded for each obesity patient: gender, age, BMI, fasting sugar, hemoglobin A1C (HbA1c), estimated average glucose(eAG), high-density lipoprotein-cholesterol (HDL-c), very low-density lipoprotein (VLDL), low-density lipoprotein-cholesterol (LDL-c), total cholesterol, triglyceride (TG) leptin, insulin, and adiponectin. The homeostatic model assessment for insulin resistance (HOMA-IR) was used to represent insulin resistance.The ADRB2 gene is a vital lipolysis receptor on human fat cells. Arg16Gly rs1042713) polymorphism of ADRB2 was significantly associated with obesity in female adolescents in Taiwan, and Gln27Glu (rs1042714) polymorphism of ADRB2 may contribute to the risk of obesity and predict obesity-related metabolic traits such as BMI, triglyceride, and systolic blood pressure in Taiwanese subjects , 22. In 13 polymoAll subjects also had physical examination analyses in this study. Bodyweight and height were measured with subjects dressed in light clothing without socks and shoes. Weight was measured  with a digital balance scale. Height was measured  with an electronic height measuring instrument. Body mass index (BMI) was calculated in the way that bodyweight was divided by body height squared. Waist circumference (WC) and hip circumference (HC) were measured to the nearest 0.1 cm using a standard tape. Waist circumference (WC) was measured between the lowest rib and the superior border of the iliac crest. Hip circumference (HC) was measured as the maximum circumference around the buttocks. Systolic and diastolic blood pressure was measured in the seated position with and an appropriately sized cuff on the left arm.In this study, genomic DNA was extracted from 3 mL peripheral blood samples of 50 individuals using the QIAamp DNA Blood Mini Kit according to the protocol recommended by the manufacturer . The quantity and quality were determined using a NanoDrop . The obesity-related loci selected in this study were associated with BMI or energy intake in previously published genome-wide and candidate gene association studies . All sixt-test was used for continuous variables, and χ2 statistics were used for categorical data. All statistics were calculated by STATA , and the statistics ≤0.05 were regarded as significant results.Demographic features, including physical and biochemical profiles, were presented either by the mean and standard deviation for continuous covariates or percentage for categorical data. To compare the differences within three different TCMBC groups, Student's 2. Obesity patients tended to have YinDC , followed by YangDC  and PSC . Moreover, combined TCMBC was quite commonly seen in enrolled patients. More than half of enrolled subjects have combined TCMBC . Eleven subjects  had balanced TCMBC. The most common comorbidities of patients in this experiment were allergic rhinitis  and hypertension .A total of 50 obese subjects were assessed for eligibility from January 1, 2018, to December 31, 2019. After a detailed explanation of the study protocol and a full period of consideration, all patients provided informed consent. Patient characteristics, such as demographic features, TCMBC, and comorbidities data, are presented in P=0.003) and YinDC  in gender. In terms of food preferences, there is a statistical difference in the intake of icy food  and deep-fried food  in PSC. YinDC has a statistical difference in alcohol consumption  and diabetes mellitus .In P=0.020) and YinDC , and we found YangDC (29.7 ± 24.8 ng/mL) has a higher leptin value than PSC (25.9 ± 21.4 ng/mL). Insulin has a statistically different trend in YinDC . On the other hand, blood sugar levels, lipid profiles, insulin resistance, and adiponectin were similar among different TCMBC groups.In the biochemical test, as given in P=0.091). PSC tended to associate with the high mutation rate of the UCP1 gene . Otherwise, FTO, GNB3, and ADRB2 had no differences among the three TCMBC groups.Tables Our study found that leptin was related to YangDC and YinDC, and there was a trend between ADRB3 polymorphism rs4994 and YangDC, while there was also a similar correlation between UCP1 polymorphism rs1800592 and PSC. To the best of our knowledge, this is the first study addressing the correlations between constitution types and polymorphisms, which might help ameliorate the advances of precision TCM. TCMBC could indicate individuals' overall health status, and an unbalanced body constitution often represents deteriorated health status, a condition that Western medicine will diagnose as no disease. With objective biochemical and genomic profiles, TCMBC may help classify the obesity population in more detail as part of personalized medicine.Most interestingly, we found that the leptin level was higher among YangDC and YinDC when compared to non-YangDC and non-YinDC subjects, respectively. The overall values of age, BMI, and high leptin were similar to other studies about the obesity population, and these facts proved the need for other classification systems. A study on the sub-Saharan African population showed that patients were about 50–55 years old, for males, the average value of BMI was 25.3 and leptin was 4.8, and for females, the average value of BMI was 28 and leptin was 18.5 . AnotherLeptin is an adipocyte-secreted hormone that circulates primarily at levels proportional to the amount of adipose tissue, signaling long-term energy storage, and secondarily at levels modified by acute changes in caloric intake . Leptin Additionally, we found the most common TCMBC was YinDC, although the patient number of YinDC was close to the patient number of YangDC and PSC. Li et al. reported the significant correlations between yang deficiency and phlegm-stasis groups with overweight and obesity outcomes with a different TCMBC questionnaire . A studyβ3-adrenoceptor (i.e. Try64Arg variant), are also associated with obesity. ADRB3 stimulates the mobilization of lipids from the white fat cell and increases thermogenesis in the brown fat cell. Decreased function of ADRB3 in white adipose tissue could slow lipolysis and cause the retention of lipids in fat cells [Moreover, we found the expression of genomic profile tended to be different among these three TCMBC. Genetic variations in the ADRB3, the at cells . YangDC at cells . UCP1 plat cells , 50. PSCat cells . Once phat cells .This study reported the relationship between TCMBC, leptin, and obesity-related SNPs. This study used biochemical parameters and genomic profiles to access TCMBC for bridging the gap between TCM theory and obesity polymorphisms. Despite its contributions, this study has several limitations. First, in our study, the patients were relatively young, and only one medical center was involved, and therefore, the generalizability may be concerned, especially the geolocation may be influential for determining TCMBC. Second, we used BCQ for assessing TCMBC, which is convenient to use and well-validated in Taiwan but has fewer constitution classifications than other studies about TCMBC –55. ThirWe found that the obese patients tended to have YinDC, while leptin is related to YangDC and YinDC, but not related to PSC. There is a trend towards near-statistical differences between the ADRB3 gene and YangDC, UCP1 gene, and PSC, and more research is needed to explain this relationship.


Mastomys natalensis are a ubiquitous and often dominant rodent across sub-Saharan Africa. Importantly, they are a natural reservoir for microbial pathogens including Lassa virus (LASV), the etiological agent of Lassa fever in humans. Lassa-infected rodents have been documented across West Africa and coincide with regions where annual outbreaks occur. Zoonotic transmission to humans most often occurs directly from infected rodents. Little is known about LASV infection kinetics and transmissibility in M.natalensis, primarily due to available animals. Here, we describe the establishment of a laboratory breeding colony of genetically confirmed M.natalensis from wild-captured rodents. This colony will provide a convenient source of animals to study LASV and other emerging pathogens that utilize M. natalensis in their enzootic lifecycles. Mastomys (family Muridae) currently contains seven or eight species of rodents [Mastomys rodents is somewhat complicated. Based on appearance, they have been referred to as rats, mice, and Praomys, a closely related African rodent, earlier in taxonomic reports [Mastomys is a unique genus within the Muridae family and remains a close relative of Praomys spp. rodents [The genus  rodents . The tax reports . The adv rodents .Mastomys rodents are ubiquitous across sub-Saharan Africa, many of which live sympatrically and are nearly indistinguishable by appearance alone . Their omnivorous and commensal habits put them in direct contact with human populations, making them an important rodent species to study as a rodent reservoir for emerging pathogens.ce alone . For exaly white   was discovered on the Jos plateau in Nigeria [M. natalensis was established as was the endemic region (West Africa) where infections in humans were occurring annually [M. natalensis. Although M. natalensis rodents have been colonized and utilized as feeder rats for herpetologists, genetic analyses have shown these animals are M. coucha. While these animals have shown utility for certain cancer, autoimmune, and infectious disease models [Approximately 50 years ago, Lassa virus , a rural village in southern Mali, approximately a 1 h drive from Bamako  were kept to establish the colony; all others were sampled as a part of ongoing surveillance programs and humanly euthanized. Sampling included the collection of a blood sample and a small punch biopsy from the ear pinnae for cytochrome b sequence analysis as previously described [The founders of the m Bamako . This siescribed . Given tPotential founders were transported to the University of Sciences, Techniques and Technologies of Bamako Medical campus located at Point G and maintained for 3–5 days in static filtered caging units, after which they were transferred to a small animal breeding facility in Bamako . Blood samples were repeatedly collected from all animals for routine molecular and serological LASV testing.The founder stock of the Rocky Mountain Laboratories (RML) colony was transferred from Bamako, Mali to RML in Hamilton Montana, according to national and international (IATA) regulations. Export out of Mali required a veterinary health certificate provided by a licensed veterinarian in Mali and a Malian export permit. Entry into the United States required permits from the Centers for Disease Control and Prevention and a Montana State exotic animal agreement, as well as a United States Fish and Wildlife Service inspection. All permits were issued in 2013 and are available upon request.M. natalensis were maintained in breeding pairs in individually ventilated cages , with autoclaved bedding , and provided with ab libitum rodent chow  and reverse osmosis water. Enrichment included nesting material  and shelters . For the first several months in captivity, animals were lightly sedated weekly with inhalational isoflurane while in their cages for health inspections and cage changing.After importation, ed cages . AnimalsMycoplasma pulmonis, pinworms, and ectoparasites.Sentinel testing at RML was conducted using standard dirty-bedding sentinels. Briefly, following a cage change, pathogen-free ICR mice were exposed to the dirty bedding for 3–4 weeks. Post-exposure, serum samples were collected from sentinel mice and submitted to an external laboratory for murine pathogen testing. Pathogens tested for included: ectromelia virus, mouse rotavirus, lymphocytic choriomeningitis virus, mouse adenovirus, Sendai virus, mouse hepatitis virus, minute mouse virus, mouse parvovirus, mouse polyoma virus, mouse norovirus, Theiler’s murine encephalomyelitis virus, M. natalensis pathogens, random animals from the colony were selected, euthanized, and a complete necropsy and examination was conducted by a certified veterinary pathologist to look for potential signs of infection or disease.Initially, sentinel testing was conducted frequently; however, after the colony was established it was reduced to four times per annum or immediately prior to a large experiment. Because of the uncertainty of potentially biased negative results due to host susceptibility of ICR mice to endogenous M. natalensis to infection with LASV, a pilot study was conducted consisting of a single group of six rodents  inoculated with 8.3 × 103 50% tissue culture infectious doses (TCID50) of LASV Soromba-R via the intraperitoneal (i.p.) route [To initially assess the susceptibility of the lab-reared .) route . On day M. natalensis. The founder stock was intended to be shipped to RML approximately 2 months after field collections; however, due to sudden political instability in Mali, the shipment was significantly delayed resulting in litters born in Mali. At the time of transfer, approximately 18 months after the original breeders were captured, a total of 90 rodents were in captivity in Mali and shipped to RML according to US federal and international regulations to expand the colony and for subsequent experiments. Based on the repeated negative test results for LASV, in addition to consistent negative results for LASV in previous rodents collected in the community where the founders were trapped, no further LASV testing was conducted prior to shipment to RML.Over the course of 4 weeks in Bamako, founder animals repeatedly tested negative for LASV infection by serological and molecular methodologies. Genetic testing  confirmed all 20 captive rodents  were Mycoplasma pulmonis. Additionally, no evidence of pinworms was noted. If the presence of ectoparasites was suspected, food supplemented with Ivermectin replaced regular mouse chow. Necropsies performed by a certified veterinary pathologist on a small subset of rodents, both directly shipped from Mali as well as laboratory-reared animals from RML, did not document any irregularities.Serological testing did not find evidence of ectromelia virus, mouse rotavirus, lymphocytic choriomeningitis virus, mouse adenovirus, Sendai virus, mouse hepatitis virus, minute mouse virus, mouse parvovirus, mouse polyoma virus, mouse norovirus, Theiler’s murine encephalomyelitis virus, or M. natalensis proved to be more difficult to handle than routine laboratory rodents. Due to their speed and agility, strict protocols were established to handle these animals, which included sealing off the room to prevent escape or loss and lightly sedating animals while in their cages with inhalational isoflurane prior to cage changes and hands-on health checks. Additionally, although the rodents were not overly aggressive with animal caretakers, bite-proof Kevlar gloves were worn whenever handling the rodents, even when under sedation.Not surprisingly, the Aggressive behavior between rodents was not common, though it should be noted that non-litter mates of the same sex were not socially housed together. Overall, the majority of aggressive events observed were in breeding pairs and most commonly consisted of female aggression toward potential or established male suitors. Cannibalism was also infrequent with only three incidents, two of which were stillborn births, observed during the first 12 months of breeding at RML.M. natalensis breeding program, 166 litters were born comprising 1618  pups. The average litter size was 9 pups, with a range of 2 to 16 per litter. In general, smaller litters were noted in the first pregnancy of younger breeders, with subsequent litters from these animals tending to trend towards the colony average. Compared with other rodent colonies maintained at RML, M. natalensis tended to reach sexual maturity and breeding potential later in life, on average around 4 months of age. Courting time for breeding pairs varied as well, with approximately one third of pairs mating almost immediately, while two-thirds taking up to 8 weeks before pregnancy was achieved. Nevertheless, the overwhelming majority of breeding pairs set up at RML (>98%) resulted in offspring. Overall, there was a very low incidence of stillbirths, with only two of the 166 litters in the first 12 months containing small numbers of dead pups. Both events occurred in the first litter of new (younger) breeders and no subsequent issues were noted in either breeding pair.Due to the delay in shipping the founder stock, 10 breeding pairs were set up from the original 20 rodents. In total, 90% of the initial pairs were fertile, resulting in the 90 rodents that were transferred to RML. Over the course of the first 12 months of the RML Both parents participated in the care of the pups. The dam did the majority of the work; however, the sire cared for the young while the dam fed and groomed. The preference for the dam and sire to remain together while raising the young often led to secondary litters being born while previous litters were still being cared for. These so called “litters on litters” were common, though they did not appear to be a problem for the parental units to maintain. In general, the sire occupied the older pups while the dam nursed the infant pups. Due to this phenomenon, colony-reared pups tended to be weaned between 16 and 20 days old since the average gestation period was estimated at 19–21 days.While not addressed in the RML colony breeding documents, there appeared to be no adverse effects on the number of pregnancies or the number of pups per pregnancy associated with the age of the dam. Similarly, longevity and life span of the animals were not addressed in these studies, although rodents exceeding 1.5 years in captivity were documented.M. natalensis had no effect on body weight during the 10-day pilot study. The majority of organ specimens collected at 10 dpi from experimentally infected animals demonstrated readily detectable levels of LASV RNA  but was also imperative in the formulation of a leading theory on the pathogenesis of SNV in humans, specifically a lack of T regulatory cell responses in cases of HPS compared to strong T regulatory responses in deer mice [Approximately 60% of emerging pathogens are of zoonotic origin and, given the diversity of the order Rodentia (≥2400 species including over 1600 murid rodents) , many ofr of SNV . The Pereer mice ,19. The eer mice . All of M. natalensis play in the maintenance cycle of an African relapsing fever spirochete [M. natalensis are susceptible to LASV infection with systemic spread of the virus. Additionally, positive viral swabs suggest the utility of this colony for studying transmission of LASV directly from rodent reservoirs. On-going efforts are now focused on long-term LASV/host infection dynamics and transmission. Given the prolific breeding potential of the laboratory colony, it is well suited to expand and facilitate several studies per year, not only on LASV but also on other potential rodent-borne human pathogens of concern from across sub-Saharan Africa.Here we sought to do similar work, albeit with an African rodent species, for the study of LASV. The colony is now firmly established and has already proved useful in studies on the ecological role irochete . The pil


SARS-CoV-2, the virus that causes COVID-19, has been detected on foods and food packaging and the virus can infect oral cavity and intestinal cells, suggesting that infection could potentially occur following ingestion of virus-contaminated foods. To determine the relative risk of infection from different types of foods, we assessed survival of SARS-CoV-2 on refrigerated ready-to-eat deli items, fresh produce, and meats (including seafood). Deli items and meats with high protein, fat, and moisture maintained infectivity of SARS-CoV-2 for up to 21 days. However, processed meat, such as salami, and some fresh produce exhibited antiviral effects. SARS-CoV-2 also remained infectious in ground beef cooked rare or medium, but not well-done. Although infectious SARS-CoV-2 was inactivated on the foods over time, viral RNA was not degraded in similar trends, regardless of food type; thus, PCR-based assays for detection of pathogens on foods only indicate the presence of viral RNA, but do not correlate with presence or quantity of infectious virus. The survival and high recovery of SARS-CoV-2 on certain foods support the possibility that food contaminated with SARS-CoV-2 could potentially be a source of infection, highlighting the importance of proper food handling and cooking to inactivate any contaminating virus prior to consumption. Coronaviridae, genus Betacoronavirus [Since the coronavirus disease 2019 (COVID-19) pandemic began in late 2019, more than 341 million cases and 5.5 million deaths have been reported worldwide, as of January 2022 [onavirus ,7. HowevIn addition to respiratory disease, COVID-19 patients also develop gastrointestinal symptoms, including nausea, vomiting, abdominal pain, and diarrhea ,9,10. ApThe World Health Organization (WHO), US Food and Drug Administration (FDA), and European Food Safety Authority (EFSA) have issued statements that food is unlikely to be associated with transmission of SARS-CoV-2 ,24,25. HA complete understanding of transmission routes for any pathogen is essential to guide the development of appropriate public health policies and control measures to prevent, or at least reduce, the risk of infection and spread within the population. Given the mounting evidence that SARS-CoV-2 can infect cells of the oral cavity and GI tract, one must consider the possibility that infection can potentially occur through ingestion of virus-contaminated foods. Since some types of food may present a greater risk of infection than others, we sought to determine the relative risks of infection from various types of foods. We previously developed and validated methods to effectively recover infectious enveloped viruses from the surface of different types of food and investigate the survival of SARS-CoV-2 on a limited number of foods over a 24 h period of time  . Others SARS-CoV-2 , obtained from BEI Resources , was propagated and titrated on Vero-E6 cells (ATCC CRL-1586). First passage virus stock was thawed on ice and serially diluted tenfold in Dulbecco’s Modified Eagle Medium  for preparation of inocula.2 pieces and placed into sterile 12-well tissue culture plates for inoculation with virus. Approximately 1.7 g of ground beef, ground pork, and plant-based meat alternative were also placed into 12-well tissue culture plates for inoculation. To best mimic a real contamination of fresh produce during transportation or handling, whole grapes and cherry tomatoes were placed into screw-top specimen containers for inoculation. Both avocado shell and pulp were included in food samples, since the avocado shell is the standard presentation in the grocery store and avocado pulp is typically consumed raw.Food samples were prepared based on the methods developed in our previous studies . Fresh f4 PFU (20 µL) of SARS-CoV-2 in multiple small droplets (<1 to 2 µL per droplet) on the food surfaces, to best mimic deposition of virus contained in droplets from a cough or sneeze. To assess maximum virus recovery from each food type, the virus was recovered immediately after inoculation (0 h) by adding 1 mL DMEM and rinsing the food surface five times by pipette. Additional food samples were inoculated and stored at refrigeration temperature (4 °C) in sealed containers to prevent desiccation. At the designated time points (1 hour (1 h), 24 h, 7 days (7 d), 14 d, and 21 d post-inoculation), the virus was recovered by rinsing with 1 mL of DMEM five times using a pipette to maximize virus recovery. Rinse fluid was collected in microcentrifuge tubes and stored at –80 °C until titer was quantified by plaque assay. For negative control samples, uninoculated food samples were rinsed and stored using the same methods.Virus inoculation and recovery were performed using methods similar to those developed in our previous studies , as we d® bag  with 1 × 104 PFU/g of SARS-CoV-2. The inoculated ground beef was massaged by hand in the sealed bag for 2 min to thoroughly mix the virus into the matrix of the meat, placed into a 4 °C refrigerator for 10 min to allow for virus spreading within the meat, followed by dividing and forming the ground beef into 1.25 cm thick patties. Ground beef patties were then cooked on a clamshell-style electric grill  to an internal temperature of 125 °F/51.2 °C (rare), 145 °F/62.5 °C (medium), or 160 °F/71.1 °C (well-done). Internal temperature of the beef patties was monitored via a Habor® digital thermometer . When the designated temperature was reached, cooking time was recorded for each cooked sample and beef patties were immediately cooled by placing the patty into a Whirl-Pak® bag and adding 20 mL of pre-chilled DMEM to prevent further cooking, followed by a thorough massage by hand to detach any virus that may have bound to the beef patties into the media. The rinse media  from each Whirl-Pak® bag was transferred into microcentrifuge tubes and stored at −80 °C until titration by standard plaque assay. As a negative control, uninoculated food samples were processed identically.Ground beef (25 g) was inoculated in a Whirl-Pak2 and then inoculum/dilutions were replaced with an agarose overlay consisting of DMEM supplemented with 8% Fetal Bovine Serum , 1% penicillin-streptomycin , and 0.5% molecular grade agarose . Back-titration of the inoculum (in triplicate) was also performed to verify the inoculum concentration. Infected plates with agarose overlay were incubated for 48 h at 37 °C with 5% CO2, and then fixed with 37% formaldehyde and stained with plaque dye. Concentration of infectious virus was enumerated by plaque counts, and the final concentrations were expressed as plaque-forming units per mL (PFU/mL).A standard plaque assay was performed on each sample. Virus samples were serially diluted tenfold with DMEM. Diluted virus samples were inoculated onto confluent Vero E6 monolayer cells in 24-well plates in duplicate. The infected plates were incubated for 1 h at 37 °C with 5% COTM RNA microPrep kit . A NanoDrop 2000 spectrophotometer  was used to measure concentrations and purity of the extracted viral RNA using reading absorbances at 260 nm and 280 nm. To determine RNA genome copy number, 10 µL qRT-PCR reactions were prepared using the iTaq Universal Probe One-Step Kit , mixed with primers and probes specific for SARS-CoV-2 N protein, and then amplified on a ViiA 7 Real-Time PCR System  [To extract SARS-CoV-2 RNA, 200 µL of food rinsing fluid from each food sample was mixed with an equal volume of TRI Reagent LS . Viral RNA was extracted and purified using Direct-zolCA, USA) . The stap < 0.05.All experiments were conducted three times in duplicate. Plaque assay data were converted to log PFU/mL prior to statistical analysis. Statistical analysis was performed using SAS version 9.4  using analysis of variance via the GLIMMIX procedure. Significant differences between means were determined based on the calculations of least square means at the level of 4 copies of SARS-CoV-2 in sputum during a typical cough, which could be deposited on the surface of foods during processing or handling [4 PFU in multiple droplets. The virus was recovered immediately after inoculation (0 h) to quantify the maximum recoverable virus from each food, and after five incubation times  at refrigeration temperature (4 °C), using a rinsing method for virus recovery that we previously validated for recovery of enveloped virus from food surfaces [COVID-19 patients have been reported to generate 1.7 × 10handling . Therefosurfaces . We assep < 0.05), infectious virus could still be recovered from the turkey at 21 dpi (1.0 log PFU/mL) (p < 0.05), and continued to decline over time. However, similar to turkey, a viable virus was still recovered at 21 dpi (1.5 log PFU/mL) (p < 0.05) and to below the level of detection (0.7 log PFU/mL) by 14 dpi , including roasted turkey representing minimally seasoned cooked meats, Swiss cheese representing cheeses in general, and salami representing a more processed and seasoned type of meat. We did not observe a significant reduction in virus titer recovered from turkey over the first 24 h . Althoug PFU/mL) . Virus c PFU/mL) . From say 14 dpi .We selected produce items that are typically consumed raw, including tomatoes, grapes, and avocados. To avoid any effects from the pulp or juice, whole cherry tomatoes and whole grapes were assessed for virus survival by inoculating SARS-CoV-2 on the surface of these fruits. Since avocados are cut to remove the pulp, which may introduce contamination from the avocado shell into the pulp, we assessed both outer avocado shell and avocado pulp for survival of the virus.p > 0.05), but infectious virus was significantly reduced by 14 dpi  (p < 0.05). However, infectious virus then remained relatively constant for 24 h. By 7 dpi, the virus was below the level of detection and completely undetectable by 14 dpi. In comparison, from the avocado pulp, the recovery of infectious virus was significantly reduced within 1 h and below the level of detection by 7 dpi (p < 0.05)  . Nonethe < 0.05) .For meats, we selected meat cuts (beef steak and pork chop), as well as ground meats (beef and pork). We also tested a plant-based meat alternative since we have observed variable results in virus survival from fresh produce (in this study and previously) . AlthougIn general, the various meats supported the survival of SARS-CoV-2 for more than two weeks at 4 °C . AlthougGround pork and plant-based meat alternatives are typically cooked well-done prior to consumption. However, ground beef formed into patties are commonly cooked to internal temperatures for rare (125 °F/51.2 °C), medium (145 °F/62.5 °C), or well-done (160 °F/71.1 °C) hamburgers based on the consumer’s preference. To determine if these internal temperatures could heat-inactivate SARS-CoV-2 in the matrix of ground beef formed into patties, we inoculated raw ground beef with the virus, formed 1.25 cm thick patties, and cooked them to internal temperatures for rare, medium, and well-done. Cooking times in a clamshell-style electric grill were approximately 1 min, 1.5 min, and 2 min for rare, medium, and well-done, respectively. Although no viable virus was detected in well-done beef patties, infectious virus was recovered from patties cooked to internal temperatures for rare (2.5 log PFU/mL) and medium (0.9 log PFU/mL), indicating that consuming rare and medium ground beef burgers may have potential risks of infection .Pathogens are commonly detected and identified on foods and food packaging by swab collection, nucleic acid extraction, and PCR-based assays. To assess the relationship between infectious virus titer and viral genome copies for SARS-CoV-2 following virus recovery, eight of the foods were selected to represent the three food categories  and qRT-PCR assay was conducted to determine the viral genome copy number . For eacIn general, at 0 h the SARS-CoV-2 RNA copy number B is 2–3 One of the most critical aspects of any pathogen is its ability to transmit and spread among populations . EffectiFollowing infection, COVID-19 patients may be asymptomatic or present with an array of variable symptoms. In addition to respiratory symptoms, gastrointestinal (GI) manifestations are common, including abdominal pain, vomiting, nausea, and diarrhea, sometimes prior to or independent of respiratory symptoms ,38,39. TIn general, high-protein unprocessed and minimally processed foods  and foods high in both protein and fats  supported viable SARS-CoV-2 for at least 14 days at refrigeration temperature. In addition, foods with higher moisture content prolonged infectivity of SARS-CoV-2, likely by preventing their desiccation and inactivation. These findings concur with other studies reporting similar results ,29. SARSAlthough SARS-CoV-2 remained infectious on deli turkey and cheese for 21 days, virus concentration on hard salami was significantly reduced within 24 h post-inoculation. Hard salami is a dry-fermented sausage with a final pH of 4.8–5.3 (0.5–1% lactic acid) and with a moisture: protein ratio less than 2.3:1 in the end product . AlthougAs we reported previously, fresh produce has variable effects on SARS-CoV-2 . In our Compared to tomatoes and grapes, we recovered a less viable virus from both the outside (shell) and inside (pulp) of avocados. Additionally, initial virus recovery (0 h) from the avocado shell was substantially less than the other produce. The avocados used in these studies were purchased from the local grocery store and avocados are often coated by commercial antimicrobial sprays, potentially contributing to the lower initial virus recovery. In contrast to tomatoes and grapes, avocado pulp exhibited a strong antiviral effect against SARS-CoV-2, reducing infectious virus to less than 1 log PFU/mL within 24 h. Extracts of avocado pulp exhibit antiviral activity against dengue virus by inhibiting viral replication, causing an increased survival rate among mice infected with the virus . Other sIn the food industry, the gold standard for foodborne pathogen detection is based on rapid PCR screening . For bacTaken together with our previous study , we asse


A popular strategy for mitigating climate change is to persuade or incentivize individuals to limit behaviors associated with high greenhouse gas emissions. In this study, adults in the mid-Atlantic United States bid in an auction to receive compensation for eliminating beef consumption or limiting vehicle use. The auction incentivized participants to reveal their true costs of accepting these limits for periods ranging from one week to one year. Compliance with the conditions of the auction was confirmed via a random field audit of the behavioral changes. The estimated median abatement costs were greater than $600 per tCO2e for beef consumption and $1,300 per tCO2e for vehicle use, values much higher than the price of carbon offsets and most estimates of the social cost of carbon. Although these values may decline over time with experience or broader social adoption, they imply that policies that encourage innovations to reduce the costs of behavior change, such as meat alternatives or emission-free vehicles, may be a more fruitful than those that limit beef consumption or vehicle use. In a new book, Bill Gates advises the public about various sources of greenhouse gases (GHGs) emissions and how changes in consumption can help achieve zero emissions and limit anthropogenic climate change . Gates rboth high-emission choices and consumption behaviors that can be adopted and sustained by broad groups of society.While fewer high-emission choices by just one individual would have no measurable impact on GHG emissions, fewer high-emission choices by many could have a large impact. Yet coordination across large portions of society is unlikely, unless changes in consumption are highly visible to others and there is a high level of in-group trust that others will be reciprocators . It follThe McKinsey Marginal Abatement Cost Curve was developed to communicate the relative abatement potential and cost associated with possible behavioral changes so government and industry decision-makers could prioritize efficient, cost-effective strategies to reduce GHG emissions . The conplasticity should be considered along with abatement potential. Plasticity is a construct used by academics to measure the proportion of nonadopters willing to change a choice or behavior ? (2) What is the lowest dollar amount for which you would be willing to limit driving your personal vehicle(s) to no more than 200  miles for one week ?” The order in which the questions were presented was randomized across participants to reduce any order effects associated with their offers. Each participant submitted ten independent offers .Compensation for the participants who ‘won’ was determined using a second-price auction, which provides incentive compatibility because participants who offer a value different from their true reservation price  are penalized . In a seThe consent form provided to participants prior to the auction communicated that by consenting to take part in the study they also agree to complete an audit for the behavioral changes if selected as a ‘winner,’ and participants were informed that a ‘winner’ of the auction would be determined from one of the five time-horizons randomly selected by the research team. The audit included providing two urine samples to a local diagnostics lab and provide two photos of their car and odometer. After consenting to take part in the study, participants were asked to sign an affidavit to comply with the audit. An image of the affidavit is shown in The auction compliance was enforced to minimize the hypothetical bias associated with eliciting values from individuals. Nevertheless, if participants with high reservation costs believed there was little chance of winning the auction, hypothetical bias might be a concern. However, no evidence of hypothetical bias was found in a recent meta-analysis of comparisons of real versus hypothetical willingness-to-accept elicitation procedures .2e) per kg of carcass weight [2e in 2018.A recent life cycle assessment of US beef estimated that direct GHG emissions are 21.3 kg of carbon dioxide emissions . In the study, we increased the overall reduction slightly from 10,373 to 10,500 miles to make it easier for participants to envision changes in their driving. The values used to determine the mileage caps are shown in S7 Table in According to the US Department of Transportation, in 2017 the average number of vehicle miles traveled per capita annually in the mid-Atlantic state where the study was conducted was 11,657 and the average annual miles driven per capita nationwide was 13,476 , 34. The2e . Using aOur convenience sample included 626 participants recruited at a public event or while visiting a retail outlet at a large public university in the mid-Atlantic region of the US. Note that, in a review of behavioral studies related to climate change mitigation, none of the meat consumption studies had more than 500 participants and over 80% of the studies had fewer than 100 participants . To qualWhile most of the participants came from the mid-Atlantic states of Delaware, Maryland, New Jersey, New York, Pennsylvania, and Virginia, the sample also include some participants from Arizona, California, Colorado, Connecticut, Florida, Indiana, Kentucky, Louisiana, Massachusetts, Michigan, North Carolina, Vermont, and Washington. The median age category of participants was 25–34, the median income category was US$40,000-$59,999, and 59% of participants were female. More detailed information about participant characteristics is reported in S8 Table in Beyond the typical socio-demographic variables, participants were also asked questions about stated consumption, attitudes towards climate change in general, and attitudes towards the links between climate change and beef and fuel consumption. A description of response options and frequency distributions for these questions are reported in S9 Table in Pearson correlation coefficients, and associated p-values testing the null hypothesis of no linear relationship between the covariates, are presented in S10-S12 Tables in 2e via reducing beef consumption is equal to the costs of mitigation per tCO2e via limiting miles traveled. Participants provided offers for both behaviors, thus we can estimate within-subject differences in costs of mitigation. Matched data provide relatively more power than unmatched data because of reduction in the variation of unobservable characteristics across individuals. A sensitivity power analysis for a two-tail Wilcoxon signed-rank mean test using matched data with type I probability and power equal to 0.05 and 0.80, respectively, and our sample size  can detect a very small effect size .The null hypothesis is that the costs of mitigation per tCOTo test the null hypothesis, we used two non-parametric tests: the Kolmogorov-Smirnov and Wilcoxon signed-rank tests. The Kolmogorov-Smirnov test was used to test the null hypothesis that the two samples are from the same distribution. While this test is informative about the overall distribution of estimated costs of mitigation, it does not incorporate the paired nature of how costs of mitigation for both behaviors were elicited. Thus, the Wilcoxon signed-rank test, a non-parametric approach, that accounts for repeated measures was also used to test the null hypothesis that the sample distributions are equal. Since costs of mitigation values are collected for five separate time horizons, it is necessary to conduct five Kolmogorov-Smirnov and Wilcoxon signed-rank tests . A Bonferroni correction was used to account for the multiple comparisons between the behaviors across the five time-horizons.2e are the same across time horizons for each behavior . A Bonferroni correction was used again to account for the multiple comparisons between the time horizons within a behavior.The Kolmogorov-Smirnov and Wilcoxon signed-rank tests were also used to test the null hypothesis that costs of mitigation per tCOThe tests described were estimated for the entire sample, and then again for plasticity targets of 25%, 50%, and 75%. Examining results for the varying plasticity targets provides the magnitude of abatement costs and potential for varying proportions of the sample engaged in a behavior change. The results focus on plasticity of 50% for two reasons: (1) it corresponds to the median cost in our sample, a value that allows us to minimize the influence of outlier offers and to make comparisons with published estimates of average abatement costs of other behaviors and policies; and (2) it corresponds to a reasonable policy target for achieving substantial reductions in GHG emissions. Results for plasticity targets of 25% and 75% are also presented, but in Finally, quantile regressions were estimated to examine heterogeneity in the amounts of compensation required to change a behavior across participant characteristics and additional covariates. Ordinary least squares regression models the conditional mean of the outcome variable, while quantile regression models specified conditional percentiles and is non-parametric so there are no assumptions implied by an error term . QuantilApproximately 60% of participants reported eating beef weekly and 35% of participants reported driving 10,000 to 14,999 miles per year. Most participants, 64%, responded that climate change is mostly caused by human activities and 68% responded that climate change was either ‘very important’ or ‘extremely important.’ Interestingly, when asked to assess the effectiveness of the behaviors evaluated in this study, 60% of participants reported that driving less was ‘highly effective’ and 24% reported that eating less meat was ‘highly effective.’ Yet more participants reported being ‘certainly willing’ to eat less meat to combat climate change, 34% versus 22% for driving less.2-equivalent (tCO2e) for eliminating beef consumption are lower than the costs for carbon-equivalent limits on vehicle use .The behavioral change with the most abatement potential—limiting personal vehicle use—is also the most expensive in our study population. Reducing GHG emissions via changes in beef consumption is likely more cost-effective than via changes in personal vehicle use. For the entire sample and for all time horizons, the within-subject private costs of mitigation per metric ton CO2e are similar for all time horizons .Also, we can reject the null hypothesis that the two empirical distributions for the private costs of mitigation per tCO2e over the various time horizons ranges from $642 to $798. For limiting vehicle use, the cost of mitigation per tCO2e ranges from $1,336 to $4,181. Thus, depending on the time horizon, GHG reductions from limiting vehicle use cost anywhere from roughly 1.8 to 6.5 times what equivalent reductions would cost from limiting beef consumption. In other words, for an equivalent amount of reduction in GHGs, adults in our sample required greater compensation for driving restrictions than for eliminating beef from their diets. Thus, policies targeted at reducing GHG emissions are likely more cost-effective for beef consumption compared to vehicle use.Whereas the private costs of mitigation from eliminating beef consumption are similar across time horizons, the mitigation costs from limiting vehicle use decrease with longer time horizons. The mechanisms that cause this decrease across time horizons are unclear, as are the reasons why such mechanisms would be active for limiting vehicle use but not for beef consumption. One hypothesis is that well-established habits, like the use of cars, have behavioral momentum and are more difficult for individuals to change . The hig2e.Policy-relevant plasticity targets for these behavior changes may be less than 50% , 37. Thu2e.We also estimate the costs at an ambitious plasticity target of 75%  regression coefficient estimates for all time-horizons in Tables As shown in S12 Table in For both behaviors, there are only two statistically significant covariates across all plasticity targets and time horizons: stated historical consumption and willingness to decrease consumption to combat climate change. Required compensation to eliminate beef and limited vehicle use increases with stated consumption and decreases with stated wiliness to change behavior. Coefficient estimates for stated effectiveness of driving less to combat climate change and responding that climate change is mostly caused by humans were also significant in a few columns for the vehicle use tables. Thus, socio-demographic characteristics may not be useful for identifying segments of the public who are amenable to changing behavior in response to climate change.2e to reduce GHG emissions by inducing 50% of our study sample to eliminate beef consumption, and $1,336 per tCO2e to reduce emissions by inducing them to limit their personal vehicle use. The estimated magnitudes for the longer time horizons would not change even if we were to assume that the perceived costs of complying with the audit are at their maximum possible values. For example, even if we were to assume that the offers for the one-week horizon reflect only the perceived costs of experiencing the random audit, the estimated abatement costs for the one-year horizon would change by less than 5% .Our results imply that it would cost at least $642 per tCO2e. These costs could be reduced by further lowering the target plasticity level, but lower levels would also yield far less GHG mitigation. An underlying lesson learned from the McKinsey Marginal Abatement Cost Curve [Even for a less ambitious goal of inducing 25% of the sample to eliminate beef or limit vehicle use, the costs for both behaviors would be more than $235 per tCOst Curve  is that 2e and varies based on assumptions  and politics [2e in 2018 dollars [Our estimated abatement costs are higher than most estimates of the social cost of carbon (SCC), even at low plasticity targets. The SCC captures the marginal damage cost of emitting a tCOpolitics . In the  dollars . At that dollars , which r dollars , which r2e is between $10 to $13 [2e,) and a low-carbon fuel standard . Our median abatement costs are also higher than the costs of removing atmospheric CO2 through enhanced rock weathering in the US ($160–190/tCO2e), although these estimates may not accurately reflect the risks associated with geo-engineering [Moreover, our estimated costs via changes in diet and travel are high in comparison to the estimated costs of reducing GHG through other means. For example, carbon offsets are sold to individuals and firms to fund projects that reduce GHG emissions . Their prices are thus market-based measures for the cost of abating GHG emissions. Currently, the price to offset a tCO0 to $13 –48. Our 0 to $13  that revineering . The difNevertheless, we acknowledge that our estimated costs collected from a sample the Mid-Atlantic US may not generalize to other populations or to variations in our targeted behavior changes. For example, our estimated costs may have been different if we had asked people to limit beef consumption rather than eliminate it, or if we had extended the time horizon for limiting vehicle use. Moreover, the perceived costs of these behavior changes may decline with individual experience via habituation or the discovery of substitutes . As the behaviors become more prevalent in a society, the perceived costs may also decline via changes in social norms or via innovations that reduce the cost and availability of substitutes. Lastly, there is an ongoing debate about the atmospheric lifetime of CH4 compared to CO2, which could imply that our estimated costs associated with eliminating beef may be revised downwards in the future .If, however, our study results approximately reflect how US adults perceive the costs associated with limiting beef consumption and driving, these behavior changes will not be easily achieved for a large proportion of the US population without substantial financial incentives, coercion, or major shifts in individual preferences. Alternatively, widespread behavior changes may be achievable through dramatic reductions in the private costs of behavior change. Thus, rather than try to limit current behaviors, governments may find that encouraging innovations that reduce the costs of behavior change, such as cheaper and more attractive meat alternatives and emissions-free vehicles, is a more cost-effective path forward both financially and politically. Also, institutions could use food service outlets to nudge food choice; for example, intentional designs of food options in a buffet-setting can influence the selection of meat , as can While our study uses revealed preference methods to measure the strength of preferences towards the behaviors of eating beef and driving a person vehicle, our approach does not account for substitutions that people likely make after reducing beef consumption and miles driving personal vehicle. To accurately measure the net effects on total food and travel emissions in field study would require extensive monitoring of individual behavior. Alternatively, a researcher could ask participants to forecast substitution behavior; however, this is likely an unreliable measure as substitution effects among possible substitutes  depend on changes in relative prices which would be impossible for participants to predict. Nevertheless, estimates from secondary data in the EU confirm that substitution effects will impact total food emissions , 22.Future research examining wider dietary habits, and not only beef, could provide insight into possible GHG-reducing behavior across product categories and substation effects. Furthermore, it is not clear what caused the difference in estimated abatement costs between eliminating beef consumption and limiting vehicle use to decreases over longer-term horizons. Future work could focus on identifying mechanisms that affect the persistence of a behavioral momentum for high-emission choices.S1 File(DOCX)Click here for additional data file.


Naturally occurring viral nanomaterials have gained popularity owing to their biocompatible and biodegradable nature. Plant virus nanoparticles (VNPs) can be used as nanocarriers for a number of biomedical applications. Plant VNPs are inexpensive to produce, safe to administer and efficacious as treatments. The following review describes how plant virus architecture facilitates the use of VNPs for imaging and a variety of therapeutic applications, with particular emphasis on cancer. Examples of plant viruses which have been engineered to carry drugs and diagnostic agents for specific types of cancer are provided. The drug delivery system in response to the internal conditions is known as stimuli response, recently becoming more applicable using plant viruses based VNPs. The review concludes with a perspective of the future of plant VNPs and plant virus-like particles (VLPs) in cancer research and therapy. One of the great challenges in biomedicine and cancer treatment is how to specifically target drugs to tumor cells and increase therapeutic outcomes. Virus-based nanotechnology holds great promise for the design of targeted therapeutics, by taking advantage of the natural properties of viruses ,2. RecenFor biotechnological applications, plant viruses have emerged as promising tools to be used as nanomaterials. Features which distinguish plant viruses from synthetic nanocarriers include stability, flexibility, diversity in shape and size for use in drug delivery and the nontoxic nature of plant viruses in humans. The revolutionary idea of producing virus-like particles (VLPs), which are empty capsids devoid of any genetic material and are replication-deficient, has been employed. VLPs acting as nanoparticles have the ability to encapsulate therapeutic molecules within capsids for imaging as well as for several drug delivery applications . Nanoparticles have been further improved for their performance in terms of stimuli-responsivity, as viruses are naturally adapted to respond to stimuli. The tumor microenvironment is not like normal cells in terms of having acidic pH, or changed redox potential, justifying the application of a stimulus-sensitive tumor targeting nanoparticles or imaging delivery systems . In ordeCancer is one of the most common death causing disease worldwide and it is characterized by uncontrolled rapid cell division and differentiation . The lacPlant viruses used for nanoparticles fall into the category of rod shaped, such as Tobacco mosaic virus (TMV)  and PotaIn this review, we provide a series of examples to discuss how plant virus architecture contributes to their applications in cancer diagnostics and therapy .Virus size data that is found in different databases has been obtained by transmission electron microscopy (TEM), and virus sizes and architecture differs significantly . A methoViruses are composed of outer protein shells that encapsulate the genomic material. The multiple copies of coat proteins that form the virus outer shell of viruses are collectively known as the capsid . PrimariResearchers have the choice of selecting the most appropriate system for any given application owing to the diversity of plant virus shape and size. A wide range of nanomedical applications have been demonstrated using the capsids of icosahedral viruses . Rod shaPlant virus-based NPs have received tremendous attention in recent years as providing the opportunity to target cancer cells. Tobacco mosaic virus (TMV) coat protein (CP) is one of the reported nanocarriers for a hydrophobic and insoluble peptide that targets the cancer cell receptor Neuropilin-1 (NRP1) . TMV hasTMV can be used as a carrier of peptides with therapeutic or targeting activity against cancer. Trastuzumab is a cancer cell inhibiting monoclonal antibody that uses the binding sites of human epidermal growth factor receptor 2 (HER2). Trastuzumab-binding peptides (TBP) are immunogenic in nature and capable of initiating production of HER2-inhibiting antibodies to seize the growth of HER2-carrying cancer cells. TMV particles displaying TBP have been created to activate this immunogenicity .A delivery system, PhenPt-TMV, has been reported whereby the anticancer drug phenanthriplatin was loaded into a hollow TMV carrier. It is an example of a stimuli responsive system, as the release of the drug is reported to be induced in the presence of an acidic environment .The plant pathogen Cowpea mosaic virus (CPMV) belongs to the Comovirus genus. CPMV is an icosahedral shaped virus with a diameter of approximately 27 nm. It is composed of RNA-1 and RNA-2 of 6 and 3.5 kb, respectively, packed in 60 copies each of large and small coat protein . CPMV isThe icosahedral shape of CPMV capsid can be loaded with precise drug cargos to target tumor and cancer cells. Other qualities of CPMV include intravital imaging (imaging living cells while they are in a multicellular organism) and improved permeability with a retention effect that improves tumor penetration . For in CPMV has been reported to interact with mammalian cells when administered orally or intravenously, or when a fluorescently modified form of virus is used to study intravital vascular imaging. The binding of CPMV to mammalian cells occurs through a cell-surface binding protein (CPMV-BP). Biochemical analysis of this CPMV-BP revealed the surface protein to be vimentin, a cytoskeletal protein known to modulate the interior of animal cells .The CPMV-DOX conjugate was developed using eighty molecules of the chemotherapeutic drug doxorubicin (DOX), covalently bound to carboxylates at the external surface of the CPMV nanoparticle. This drug delivery vehicle was found to be more cytotoxic than free DOX when used in low concentration, however, CPMV-DOX cytotoxicity is time-delayed at higher concentrations . Cancer As immunotherapies are resisted by tumor microenvironment, intratumoral administration of CPMV based nanoparticles as an immune stimulant has been reported to overcome the endogenous immunosuppression .Potato virus X (PVX) is a member of the family Alphaflexiviridae, genus Potexvirus, and an important plant pathogen of the family Solanaceae that specifically infects potato, tomato and tobacco ,52. AmonPVX can carry large payloads due to its flexible and filamentous structure, making it possible to use for pharmaceutical and imaging applications. Accordingly, PVX attain advanced tumor targeting and retention qualities, better than spherical nanoparticles. Qualities such as advanced pharmacokinetic profiles, low in vivo toxicity and enhanced functional properties make this protein-based nanoparticle suitable for biomedical applications .The PVX particle is 515 × 14.5 nm in dimension and comprised of 1270 subunits of CP, 8.90 ± 0.01 per turn subunits and 3.45 nm of helix . The C-tWhen filamentous PVX VLPs are heated to 70 °C they swell, and at 90 °C, spherical VLPs are formed , akin toUnlike other reported viruses, the assembly of PVX CP subunits into filamentous VLP, in vivo or in vitro, is not possible in the absence of genomic RNA. This reflects the unique connection between virus RNA and CP . PVX nanAn efficient and new drug delivery system has been reported for Non-Hodgkin’s B cell lymphomas (NHL) based on PVX binding affinity towards malignant B cells. In a mouse model when PVX was loaded with the chemotherapy monomethyl auristatin (MMAE) and effectively administered to tissues harboring malignant B cells lead to inhibition of NHL growth. Thus, PVX based nanoparticle is an efficient drug delivery platform for malignancies of B cell .Other plant VNPs are getting more attention due to their structural and functional characteristics when compared to synthetic nanoparticles. For example, the capsid proteins of plant virus Brome mosaic virus (BMV) have been reported for the synthesis of superparamagnetic nanoparticles (SPIONs) . BMV is Viral capsids of icosahedral shaped Cowpea chlorotic mottle virus (CCMV) behave as an excellent natural protein cage for encapsulation and packaging of genomic nucleic acid. These molecular containers can be used as vessels for the entrapment and packaging of synthetic cargo . PhysaliThe ricin epitope has been expressed on the surface of the icosahedral plant virus Tomato bushy stunt virus (TBSV) VLPs. Injection of these chimeric VLPs generated antisera into mice which makes TBSV a perfect choice for vaccine design .The Virus-like nanoparticles (VLPs) derived from CP of plant viruses used to treat cancer are listed in Magnetic resonance imaging (MRI) is a promising technology for the diagnosis of disease due to its high resolution and deep contrast, however, virus-based nanoparticles have been explored to overcome the drawback of low sensitivity . TMV is Noninvasive imaging systems that are for early diagnosis of cancer, such as magnetic resonance imaging (MRI) and computed tomography (CT), have some limitations ,73. NanoThe use of nanoparticles (NPs) as drug delivery carriers for the treatment of infectious and chronic diseases including cancer are advantageous when compared with naked drugs . The mosVLPs mimic the native conformation of viruses, propagate their innate immunogenicity and promote safety due to the lack of their genetic material, and therefore form an attractive platform for vaccine development ,79. FurtVLPs have been attached to TAAs (tumor associated antigens) using chemical conjugation, genetic fusion and enzyme-mediated ligation techniques. Among these various strategies, the most common method has been chemical conjugation. For example, the human epidermal growth factor receptor 2 (HER2) epitope, when conjugated to the icosahedral CPMV, was successfully delivered to the lymphatic system with enhanced uptake and activation of APCs that led to an augmented anti-HER2 immune response. The CPMV HER2 candidate vaccine slowed tumor progression and metastasis in mouse models, enhancing survival . ImportaCancer vaccines against carbohydrate antigens associated with tumors (TACAs) could be useful for diminishing tumor progression. Nevertheless, carbohydrates are weakly immunogenic and therefore, plant viruses used as carriers of these molecules could enhance the immune response to TACAs. CPMV-TACA conjugates targeting the Tn antigen   were demThe tumor microenvironment poses a great challenge to immune clearance by virtue of being immunosuppressive and favoring immune escape of the tumors through the inhibition of anti-tumor T-cells ,86. CPMVPlant virus VLPs have also been used as combination therapies to augment their immune efficacy. The PVX-DOX (doxorubicin)  combinatPresently, clinical treatment for cancer is primarily addressed by chemotherapy . NeverthPlant virus VLPs have several attractive features that make them appropriate for targeted administration of therapeutic molecules. The anti-cancer drug doxorubicin (DOX), has been successfully delivered using VNPs and VLPs. Specifically, red clover necrotic mosaic virus (RCNMV) VLPs have been used to both encapsulate DOX  as well Plant virus VNPs have been used for targeted administration of platinum-based drugs against cancer. This is important, as 50% of chemotherapy treatments involve the use of these platinum-derived drugs. TMV has been demonstrated to efficiently deliver Cisplatin  and PhenAnother anti-cancer drug, mitoxanthrone (MTO), is a topoisomerase II inhibitor and has been shown to be encapsulated by TMV  and CPMVViruses are inherently natural gene-delivery vehicles. Plant viral VLPs in particular, by virtue of being devoid of their own genetic materials, easily encapsidate nucleic acids. This makes them highly useful in delivering genes and therapeutic nucleic acids. It is reported that  67% of clinical trials involving gene therapy use VLPs and viruses as vectors for transmission of genetic materials. Tian et al., (2018) demonstrated that transacting activation transduction (TAT) peptide conjugated to the external surface of TMV augmented internalization along with increased ability to escape endo/lysosomal compartments . Most ofIn comparison to nucleic acids, proteins and polypeptides are more difficult and complex to encapsulate, thus limiting their delivery into cancer cells. This could be resolved by fusing the polypeptide to the VLP capsid protein by chemical or genetic means. This could direct cargo localization into the interior of the VLPs during the synthesis and assembly of the capsid in vivo . Viral cHelical viruses such as PVX  and TMV VNPs developed from plant viruses are non-infectious for warm-blooded mammals, comprised of only virus shells and lacking a viral genome. Genetically engineered viruses can be selected for required binding sites and modified properties . In addiThe future of plant virus nanoparticles in anti-cancer therapy will continue to evolve. The vast majority of scientific studies have taken place either in cell culture or in animal models. Thus, the next significant challenge will be the design and implementation of human clinical trials. The authors are unaware of any such trials that have been undertaken to date; however, much talk of this next step is ongoing within the scientific community, with optimistic expectations with respect to safety and efficacy. Besides this initial hurdle, delivery mechanisms for plant virus particles into patients need to be explored in more detail. Finally, a regulatory framework must be set in place to ensure that plant virus nanoparticles can safely reach the market.