Borrelia-specific antibodies in serum did not contribute to the diagnosis of Borrelia arthritis or Borrelia-associated dermatitis in a young woman with ongoing treatment with rituximab due to multiple sclerosis. The diagnosis was confirmed by the detection of Borrelia-DNA in a skin punch biopsy. The patient history did not reveal any tick exposure. She had suffered for several months from fluctuating pain and swelling of the right knee as well as skin involvement with redness and oedema around the ankle of the same leg. Monoarthritis was confirmed by a rheumatologist. Knee puncture was performed but the synovial fluid was only sufficient for microscopic examination of crystals. Neither monosodium urate crystals nor calcium pyrophosphate crystals were found. Borrelia serology in blood revealed borderline levels of immunoglobulin (Ig)M and IgG, respectively. Treatment with doxycycline resulted in resolution of the joint and skin manifestations within a month. This case highlights that Borrelia-specific antibody levels cannot be reliably interpreted in patients who have received B-cell depleting therapy. Under these circumstances, detection of the bacterial genome in different body fluids, such as in the skin, can be a useful complement to the diagnosis of Lyme disease. In this young female, the diagnosis would certainly have been further delayed without the detection of Borrelia-DNA in the skin. An atypical clinical presentation and absence of an adequate immune response to infections are common phenomena in patients with primary immunodeficiency disorders (PID)  and multneuromyelitis optica was suspected due to bilateral optical neuritis and the presence of spinal cord lesions. However, antibodies against aquaporin-4 and myelin oligodendrocyte glycoprotein were not detected and the magnetic resonance imaging (MRI) of the brain and spinal cord as well as cerebrospinal fluid (CSF) findings were supportive of MS. Apart from persistent bilateral severely reduced visual acuity she had no other signs of neurological dysfunction. She had previously been in good health and had no family history of PID, or other systemic inflammatory diseases.This case illustrates a 20-year-old female diagnosed with MS at the age of 17. She was initially treated with tocilizumab as 9/L). During this period, there were no signs of neuroinflammatory activity of MS.Eighteen months prior to the episode of arthritis and skin symptoms reported here, she was started on off-label treatment with rituximab (RTX). RTX is the most frequently used immunomodulatory drug for MS in Sweden according to the Swedish MS registry . Initialrubor, tumor, and calor accompanied by a discretely reduced range of motion. The general status was good without fever. The lower right leg was diffusely swollen and two circular erythematous areas around the ankle were seen M and IgG antibodies against recombinant Borrelia antigens . The results were interpreted to be of uncertain clinical significance. Laboratory results are detailed in Despite the vague antibody results, there were an enduring clinical suspicion of Borrelia infection. Skin biopsies from one of the erythematous areas at the ankle were performed. Standard histopathology showed mild non-specific inflammation. Borrelia-DNA was detected in the biopsy analyzed by polymerase chain reaction (PCR). The method amplifies a 116 base-pair long fragment of the 16S rRNA gene. In addition, a lumbar puncture was done, and CSF was analyzed without presence of intrathecal Borrelia-specific antibodies or elevated levels of the B-cell chemokine CXCL13. Thus, the final diagnosis was Borrelia associated dermatitis and arthritis (Lyme disease).Prior to the diagnosis of Lyme disease, the patient was prescribed topical steroids for the skin manifestations and the joint symptoms were managed with paracetamol. Once the diagnosis of Lyme disease was confirmed, doxycycline 200 mg once daily for 3 weeks was prescribed. The knee and skin symptoms dissipated during the following month.At the last follow-up 1 year after the antibiotic treatment had been ended, there was still minor swelling of the lower leg but no signs of arthritis or dermatitis. An MRI of the lower leg showed mild oedema in musculus soleus and gastrocnemius. Creatinine kinase in plasma was within normal reference.IMT in general, and particularly B-cell depleting therapies, may be associated with an increased risk of infections , 6, 7. Sacrodermatitis chronica atrophicans, the late cutaneous manifestation of borreliosis  for the publication of any potentially identifiable image or data included in this article.JS, CS, and CD had full access to all of the data and takes responsibility for the integrity, accuracy and interpretation of the data. All authors contributed to the article and approved the submitted version. All authors were involved in drafting the manuscript or revising it critically for important intellectual content and all authors approved the final version to be published.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Dengue virus (DENV) and is considered to be the most common arbovirus worldwide. The clinical characteristics of dengue may vary from asymptomatic to severe complications and severe organ impairment, particularly affecting the liver. Dengue treatment is palliative with acetaminophen (APAP), usually known as Paracetamol, being the most used drug aiming to relieve the mild symptoms of dengue. APAP is a safe and effective drug but, like dengue, can trigger the development of liver disorders. Given this scenario, it is necessary to investigate the effects of combining these two factors on hepatocyte homeostasis. Therefore, this study aimed to evaluate the molecular changes in hepatocytes resulting from the association between DENV infection and treatment with sub-toxic APAP concentrations. Using an in vitro experimental model of DENV-2 infected hepatocytes (AML-12 cells) treated with APAP, we evaluated the influence of the virus and drug association on the transcriptome of these hepatocytes by RNA sequencing (RNAseq). The virus–drug association was able to induce changes in the gene expression profile of AML-12 cells and here we highlight and explore these changes and its putative influence on biological processes for cellular homeostasis.Dengue is an acute viral disease caused by  Aedes. The disease is caused by Dengue virus (DENV), an enveloped, positive-sense single-stranded RNA virus with four distinct serotypes (DENV-1 to -4). Approximately 3.9 billion people from 128 countries live at risk of infection by DENV  iv3.38.3) . The heav3.38.3) .To identify enriched gene ontology (GO) categories ,30, the Mus musculus reference genome (version GRCm38.p5) resulted in uniquely mapped reads ranging from 80.53% to 84.24%. A wider analysis of the results was performed through a multidimensional scaling plot designed to indicate sample relationship similarities. The multidimensional scaling analysis demonstrated that the infection by DENV-2 was determinant to separate this group of samples from the untreated and APAP treated cells  in stress-induced antiviral cell response a) and wiDENV infection and APAP treatment are capable of triggering severe liver disorders ,11,35. AStudies that have elucidated the mechanism of APAP induced hepatotoxicity have significantly contributed to the development of diagnostic and treatment methods for this acute liver failure . HoweverIn this context, the analysis of transcriptomic changes in AML-12 cells resulting from treatment with sub-toxic APAP concentration was performed. The APAP induced liver injury model in mice is suitable for studying metabolic functions or immunological reactions after an APAP overdose and is a directly transferable model for the pathogenesis of human hepatotoxicity . Thus, AIt was also observed that sub-toxic APAP doses upregulated genes mainly related to the mechanisms of the cell’s multiplication. It is known that, at lower APAP doses, acute liver damage is followed by compensatory liver regeneration. Viable hepatocytes near the injured area undergo mitosis and replace dead cells, promoting tissue recovery . The oveDENV can infect different cell types causing altered clinical and pathological effects . Among tThe changes in the transcriptome in response to DENV can vary depending on different DENV strains and stages of the virus life cycle . In thisThe downregulation in genes involved in mitochondrial organization corroborates with studies that showed that DENV infection induces mitochondrial and consequently bioenergetic dysfunction, contributing to viral replication . During In DENV infected AML-12 hepatocytes, upregulated genes are mainly related to antiviral immune responses. The innate immune response against DENV is mainly induced by RIG-I and MDA5 receptors, which recognize dsRNA, detect viral replication in the cytoplasm and constitute an essential part of the innate immune response against the virus . This prAn epitome of results obtained allows the description that in AML-12 hepatocytes treated with sub-toxic APAP doses, genes involved in the mitosis process were upregulated while in cells infected with DENV, genes involved with the antiviral cellular response were upregulated. However, it is noteworthy that both in DENV infection and in APAP treatment there is a reduction in the expression levels of the genes involved with lipid metabolism. Lipid metabolism is an extremely important mechanism in several processes that guarantee cellular homeostasis . It has Lipids are stored mainly as triacylglycerols, cholesterol esters and retinyl esters in organelles composed of a phospholipid monolayer known as lipid droplets. These lipids can be accessed mainly through the lipolysis process, which involves the decomposition of triacylglycerols and esters by cytosolic lipases. Another important process for accessing cellular lipid content is known as lipophagy, a specific subset of selective autophagy that targets lipid droplets, catabolizing their components into free fatty acids and glycerol. Lipophagy is very important to lipid metabolism and the regulation of cellular homeostasis , presentTo verify the effects of the association between the virus and the drug on the transcriptome of AML-12 cells infected with DENV and treated with APAP, the transcriptome of these cells was compared with all other conditions . Downregulated genes are involved mainly in the mitotic cell cycle and upregulated genes are related to cellular component organization and intracellular signal transduction. Thus, when combined, DENV and APAP seemed to modify pathways that were not individually altered by the virus or the drug. DENV and APAP also acted synergistically, reinforcing the expression patterns of some genes and antagonistically, reversing the expression patterns of other genes. There was a reduction in the expression levels of genes involved with cell division and an increase in the expression of genes related to the apoptosis process, which is important for mitochondrial morphology and cellular bioenergetic mechanisms.The “Role of PKR in stress-induced antiviral cell response” pathway is the main canonical MetaCore pathway associated with Dengue disease culminating in processes of inflammatory, antiviral and antistress responses besides the inhibition of cell proliferation. The association between DENV and APAP decreased or reversed the expression patterns of several genes in relation to those of DENV infected cells, indicating a possible influence of this association on the responses triggered by the virus in the hepatocytes. Regarding the main MetaCore pathway associated with liver failure, the “IL-18 signaling” pathway is related to Th1 cell activation and differentiation, oxidative burst, apoptosis, cytotoxicity, immune responses, adhesion, cell migration and proliferation. IL-18 mediated signaling is essential for the host’s defense, inflammation and tissue regeneration processes, being related to the mechanisms of liver injury in vivo . The comTaken together, our results provide an overview of the effects of DENV infection, APAP treatment and the interaction between DENV infection and APAP treatment in the transcriptome profile of AML-12 hepatocytes. Both DENV infection and APAP treatment impact the expression levels of genes involved to lipid metabolism in hepatocytes, a mechanism extremely important for cell homeostasis that should be the target of further studies. It was also observed that the association between DENV and APAP interfered in the expression profile of genes that were molded only by DENV or only by APAP; thus, the joint presence of the virus and the drug led to the alteration of the expression of genes in a way that restored the profiles of expression at levels similar to those of the control (clusters 3 and 4). However, for some genes, the joint action of DENV and APAP made these profiles opposite to those observed in the control , indicating genes whose expression could act to disrupt the normal cellular metabolism. Investigating the influence of the expression levels of these genes on the cell phenotype may contribute to the understanding of the mechanisms that could induce liver injury during dengue treatment using APAP. Therefore, it may contribute significantly to the safer and efficient management of dengue patients.


Melanoma, like other solid tumors, releases DNA molecules that are referred to as circulating tumor DNA (ctDNA), into the blood and other biological fluids. ctDNA analysis performed with molecular biology techniques can provide important information on the aggressiveness of the disease and its genetic characteristics. This review aims to highlight all the possible clinical applications of ctDNA analysis that can contribute to an improvement in the diagnosis and therapy of melanoma.Malignant melanoma accounts for about 1% of all skin cancers, but it causes most of the skin cancer-related deaths. Circulating tumor DNA (ctDNA) testing is emerging as a relevant tool for the diagnosis and monitoring of cancer. The availability of highly sensitive techniques, including next generation sequencing (NGS)-based panels, has increased the fields of application of ctDNA testing. While ctDNA-based tests for the early detection of melanoma are not available yet, perioperative ctDNA analysis in patients with surgically resectable melanoma offers relevant prognostic information: i) the detection of ctDNA before surgery correlates with the extent and the aggressiveness of the disease; ii) ctDNA testing after surgery/adjuvant therapy identifies minimal residual disease; iii) testing ctDNA during the follow-up can detect a tumor recurrence, anticipating clinical/radiological progression. In patients with advanced melanoma, several studies have demonstrated that the analysis of ctDNA can better depict tumor heterogeneity and provides relevant prognostic information. In addition, ctDNA testing during treatment allows assessing the response to systemic therapy and identifying resistance mechanisms. Although validation in prospective clinical trials is needed for most of these approaches, ctDNA testing opens up new scenarios in the management of melanoma patients that could lead to improvements in the diagnosis and therapy of this disease. Melanoma is an aggressive and deadly disease that is responsible for the largest number of skin cancer-related deaths, although it comprises less than 10% of skin cancers. The high mortality rate of melanoma is due to the late diagnosis and to the highly metastatic potential of melanoma cells, which typically spread in different organs . For. For8]. cfDNA can be isolated from different body fluids. When cfDNA is obtained from the peripheral blood, plasma is preferred to serum, especially because the large amounts of wild-type DNA released by white cell lysis during clotting can cause a further dilution of ctDNA. To prevent clotting and DNase I activity, it is recommended to use ethylenediamine-tetraacetic acid (EDTA) tubes or tubes containing formaldehyde-free preservative reagents for blood collection . When EDBecause of the possible dilution of tumor DNA in normal DNA, highly sensitive methods are needed to detect variants at low mutant allelic frequency (MAF) in the cfDNA. In this regard, sensitive methodologies have been developed to detect and quantify rare variants in the blood of cancer patients, with analytical sensitivity ranging between 0.005 and 5% . These tBRAF, NRAS, KIT or NF1 [BRAF V600 variants in cfDNA, because of the availability in clinical research and clinical practice of drugs targeting tumors carrying these mutations.Melanoma most frequently harbors genomic alterations in T or NF1 ,14. In pBRAF-V600E mutation [A number of different approaches based on qPCR have been developed in order to improve its sensitivity for the analysis of rare variants. Allele-specific PCR (AS-PCR), allele-specific amplification refractory mutation system PCR (ARMS) and peptide nucleic acid (PNA)-based PCR have demonstrated, in different studies, analytical sensitivity up to 0.001% for the detection of the mutation .BRAF in the plasma from stage IIIc–IV melanoma patients, Denis et al. observed that the Therascreen BRAF RGQ kit (Qiagen) was able to detect BRAF mutations in 73.7% of plasma samples from patients with BRAF-positive tumors. Similar results were obtained with the ctBRAF Mutation Detection Kit (Entrogen), which showed a positive percent agreement (PPA) of 68.4%. In all patients with wild-type BRAF tumors, the test was negative for cfDNA with both techniques [Comparing the performance of some qPCR tests in detecting mutant chniques .BRAF/NRAS (Roche) mutation test was used to analyze the ctDNA of 68 patients with stage III or IV melanoma. The test is an allele-specific, real-time PCR assay for the qualitative detection and identification of exon 11 and 15 mutations in the BRAF gene and exon 2, 3 and 4 variants of the NRAS gene from tumor tissue or plasma samples. The expected mutations were detected in the plasma of 34/68 patients (50% sensitivity), and the results obtained by the Cobas analysis were similar to those obtained by digital PCR (dPCR) [In a recent study, the Cobas R (dPCR) . ImportaNRAS, BRAF, KRAS or EGFR mutations in either ctDNA or genomic DNA isolated from tissue. The test allows the detection of BRAF V600 mutations in plasma with up to three mutant copies per PCR reaction with an analytical sensitivity of 0.01% [Recently, Biocartis (Belgium) developed a new fully automated platform, designated Idylla, for detecting the major of 0.01% . A 75% cof 0.01% .BRAF-V600E test detected the variant in 84.3% of plasma-derived cfDNA obtained from advanced melanoma patients [of BRAF-V600E/V600K mutations in cfDNA was observed using a BEAMing-based assay [Emulsion-PCR-based methodologies, such as dPCR, droplet digital PCR (ddPCR) and BEAming  have a greater sensitivity as compared with qPCR, overcoming the problems due to the low levels of cfDNA and the low MAF of the variants ,5. In fapatients , while aed assay .Although qPCR and emulsion-PCR have a high sensitivity and specificity, both these methodologies have the limitation of being able to interrogate only a few loci for analysis. Therefore, the use of these techniques is limited to the screening of the most frequent known mutations. In addition, they cannot provide information on the genetic evolution of the tumor that might be relevant for therapeutic intervention, such as the identification of resistance mutations.Many of these limitations are overcome by NGS, which is a high-throughput screening method that allows the simultaneous analysis of multiple genes and the detection of novel alterations, including low-frequency mutations. However, sensitivity represented, for a long time, a limiting factor for using NGS to test cfDNA. In this respect, targeted-sequencing panels are more suitable compared with whole-genome sequencing (WGS) or whole-exome sequencing (WES) due to their higher sensitivity, and reduced turnaround time and cost . Novel NTargeted-sequencing panels of different sizes, from a few mutations to hundreds of genes, are available both as a service and sold by different providers for independent laboratories . DiffereThe chances of survival for melanoma patients significantly increase when the disease is diagnosed at an early clinical stage. Therefore, the improvement of melanoma early detection methods is fundamental for a proper management of this disease. However, several issues including the tumor heterogeneity, the tumor growth dynamics and the timing of metastasis, as well as the feasibility and cost of routinely applying liquid-biopsy techniques in clinical practice, make early detection challenging .BRAF V600E could represent a suitable marker for melanoma detection, this mutation was identified only in ctDNA from stage III–IV melanoma patients, whereas, in early stages, it was undetectable in the majority of the cases [BRAF-V600 mutations are present in only 50% of melanomas, while they can be found in a number of different tumor types [In a study aiming to define whether he cases . While tor types . ProspecNGS-based technologies might improve the use of cfDNA testing for the early detection of cancer. By testing a number of genetic alterations in a single analysis, these methods can significantly increase the sensitivity of the test. However, the detection of genetic alterations in the cfDNA might not have sufficient specificity for the early diagnosis of cancer. In this respect, the integration of information deriving from cfDNA analysis with other circulating biomarkers might significantly increase the sensitivity and specificity for the detection of melanoma in the early stages of development, as demonstrated for other cancer types . In addiA considerable fraction of patients with localized melanoma who undergo surgery with curative intent experience a relapse of the disease. Local recurrence and distant metastasis in melanoma patients are heavily dependent on tumor size. They occur in 50% of patients with a tumor thickness larger than 4 mm , in regiIn this scenario, perioperative ctDNA testing in patients with surgically resectable melanoma can offer different, relevant prognostic information: (i) the detection of ctDNA before surgery might correlate with the extent and the aggressiveness of the disease; (ii) ctDNA testing after surgery and, eventually, adjuvant therapy might identify MRD; (iii) testing ctDNA during the follow-up after curative resection might detect a tumor recurrence at the molecular level, thus anticipating clinical or radiological recurrence. Indeed, given the possibility of minimally invasive repeated sampling, ctDNA testing can allow real-time monitoring during the course of the disease.The detection and monitoring of MRD are widely established in patients with hematological malignancies . More reBRAF, NRAS or cKIT in tumor tissue were included in this study. ctDNA was detected in 34% of the patients in the discovery cohort and 33% in the validation cohort. The presence of ctDNA was significantly associated with tumor burden. More importantly, patients with detectable ctDNA had worse melanoma-specific survival in both the discovery cohort  and the validation cohort , and this difference was confirmed via multivariate analysis .Similar findings have been reported in patients with high-risk stage III melanoma . In partThe prognostic value of pre-operative ctDNA detection was confirmed in an additional study that enrolled stage III melanoma patients who were tested for ctDNA by dPCR . HoweverBRAF or NRAS mutation in their baseline-resected tumors. Patients with detectable ctDNA after surgery had a significantly increased risk of death compared to those with undetectable ctDNA, with HR = 2.50 for overall survival (OS) after adjustment for performance status [The prognostic value of post-operative ctDNA testing was also explored in a retrospective analysis of plasma samples from 161 stage II/III patients carrying either a e status . The afoAlthough the above-summarized findings suggest a possible prognostic role of cfDNA testing in patients with resectable melanoma, these studies have several limitations. First, they included only patients with known mutations in a limited number of genes, thus excluding those patients that carry rare variants. In addition, while the specificity of the test is high, its sensitivity is relatively low because a significant fraction of ctDNA-negative patients have recurrences of the disease. In this respect, the use of NGS-based assays might increase the sensitivity of the test, being able to detect multiple mutations at the same time. Alternatively, the NGS testing of tumor tissue could be used to identify an adequate number of variants to test for in the cfDNA with highly sensitive dPCR-based assays.The availability of different therapies makes highly relevant the use of biomarkers to stratify melanoma patients and identify the best therapeutic strategy for each individual patient. In this respect, cfDNA testing can provide relevant information at different levels: (i) the genomic profile of the disease and tumor heterogeneity, (ii) the prognosis, (iii) the response to therapy, and (iv) the development of resistance mechanisms.BRAF and NRAS mutations in plasma-derived cfDNA from patients with advanced melanoma  and the number and type of metastatic sites [The sensitivity of the cfDNA test might be affected by the localization of the tumor. In fact, patients with visceral, bone or lymph node involvement often display higher levels of ctDNA as compared with patients with extensive subcutaneous disease or brain metastases . In agreic sites .BRAF mutations [It has also been reported that the detectability of ctDNA is related to the nature of the mutated gene. Herbretau described a sensitivity of the cfDNA test of 36% for NRAS mutations and 66% for utations . In addiutations .BRAF mutations have been identified through cfDNA analysis but not in the corresponding tumor tissue [BRAF mutations in melanoma cells.The sensitivity of cfDNA testing is also limited by the frequency of the mutant alleles in the context of wild-type DNA derived from normal cells. On the other hand, in selected cases, r tissue . Such diBRAF mutations in melanoma, are often clonal but can occasionally be subclonal [BRAF mutational statuses have been found between different sites of a primary tumor (intratumor heterogeneity), between a primary tumor and metastases, and between different metastases of the same patient [BRAF and BRAF mutation not detected in tumor tissue, the testing of additional samples from a different tumor area confirmed the presence of the BRAF variant [It has been demonstrated that driver mutations, including ubclonal . This mi patient ,57. In t patient . Indeed, variant .BRAF mutational status over time as a consequence of tumor heterogeneity [BRAF mutation status with a liquid biopsy might represent a non-invasive approach to confirming the mutational status of the disease.Analysis of longitudinal samples from melanoma patients revealed that recurrent lesions might show a different ogeneity . In the BRAF-V600 mutations are the only approved biomarker in melanoma, the majority of data on genome profiling with liquid biopsies are based on the use of assays specific for these variants. However, NGS-based technologies might provide a better portrait of the genomic landscape and heterogeneity of melanoma, which might be relevant for therapeutic purposes. For example, it has been described that MITF and TP53 alterations are more frequent in patients with rapid progression of the disease following targeted therapy, while NF1 alterations are more common in cases with complete responses [Because the BRAF-V600E/K metastatic melanoma patients to evaluate the clinical activity and the safety of the BRAF inhibitor dabrafenib, a correlation was found between the basal levels of BRAF-V600E in the cfDNA and tumor burden but not for the V600K variant, as assessed by using BEAMing technology [BRAF-V600E mutation showed a lower response rate and a shorter progression free survival (PFS) when treated with dabrafenib. In line with these findings, similar results were obtained in a larger cohort of 732 patients from four clinical studies of targeted therapy in metastatic melanoma . Patients negative for BRAF mutations in the cfDNA had longer PFS and OS and higher rates of response to dabrafenib and trametinib, as compared with patients with detectable cfDNA BRAF mutations [BRAF mutations in the cfDNA was an independent predictive factor for shorter PFS in three out of four studies, and for shorter OS in one study.The identification of genetic alterations in cfDNA and the assessment of ctDNA levels before the administration of any systemic treatment may provide important prognostic information for patients with metastatic melanoma. In fact, a number of studies have demonstrated that high baseline levels of ctDNA correlate with a worse prognosis in metastatic melanoma patients treated with targeted therapy ,23,45. WBRAF-V600E copies in cfDNA from 20 patients with metastatic melanoma showed a direct relationship between the BRAF-V600E copy numbers and clinical outcomes, where basal concentrations <216 copies/mL were significantly associated with better outcomes  as compared with higher concentrations  [BRAF mutations in cfDNA and either survival or tumor responses in 25 patients receiving a combination of dabrafenib plus trametinib [BRAF inhibitor, and the response rate was only 32%. The presence of a mechanism of resistance to a targeted therapy might represent a confounding factor for the prognostic role of ctDNA.Studies in smaller cohorts of patients further confirmed these findings. The quantification with ddPCR of mutant  months) . Howeverametinib . ImportaThe basal levels of ctDNA are also a relevant prognostic marker in patients treated with immunotherapy. Seremet assessed ctDNA levels in 85 advanced melanoma patients using either the Idylla assay or a ddPCR test . They foLow baseline ctDNA levels were associated with higher response rates and longer PFS and OS in studies that included cohorts of patients treated with either targeted therapy or immunotherapy ,46. ImpoThe strong prognostic value of ctDNA levels in metastatic melanoma patients suggests that this parameter might better recapitulate the disease burden as compared with other prognostic factors. In this respect, several studies have demonstrated a correlation between ctDNA levels and the American Joint Committee on Cancer (AJCC) stage, number of metastatic sites, and serum levels of Lactate dehydrogenase (LDH) and S100 ,63. A siThe availability of predictive and prognostic biomarkers significantly improved the selection of appropriate therapeutic approaches in melanoma patients. Nevertheless, the response to both targeted therapy and immunotherapy is often heterogeneous, even in molecularly selected cohorts of patients. Therefore, the availability of tools to assess the response to therapy could further improve the therapeutic approaches for patients with metastatic melanoma. In this respect, the analysis of cfDNA could represent a non-invasive and repeatable technique for the early detection and monitoring of melanoma response and/or progression.BRAF mutation testing of cfDNA to monitor the response to BRAF inhibitors [BRAF and MEK inhibitors [BRAF-mutant melanoma. Although these studies enrolled a limited number of patients and different qPCR [BRAF mutations in cfDNA, while an increase in BRAF mutant DNA was observed at progression. Interestingly, the increase in ctDNA levels preceded the clinical progression of the disease in a significant fraction of patients, with a lead time up to 110 days [A number of studies have addressed the possibility of using the hibitors  or combihibitors  in patieent qPCR  or emulsent qPCR ,51-based110 days .BRAF mutations in cfDNA using the Idylla test was also explored in a phase II trial of a combination of dabrafenib plus trametinib in patients with advanced BRAF-V600-mutant melanoma pre-treated with targeted therapy [BRAF-V600-mutant ctDNA after 2 weeks of treatment as compared with non-responding patients. In addition, the persistent detection of BRAF-V600-mutant ctDNA after 2 weeks of therapy was correlated with a shorter PFS as compared with that for patients with undetectable ctDNA .The predictive value of monitoring the  therapy . Upon anBRAF inhibitors [Some studies have reported a very early spike in ctDNA concentration occurring 24/48 h after starting treatment with hibitors ,64. ThisBRAF mutations in the cfDNA could represent a valuable biomarker for assessing the early response to targeted therapy in melanoma patients.Taken together, these findings strongly suggest that monitoring Testing cfDNA could also provide important information for the evaluation of the response to immune-checkpoint inhibitors in melanoma and other cancers. In fact, the assessment of the response to immune therapies is sometimes difficult, due to the possible increase in the tumor lesions because of the immune reaction, which mimics a progression of the disease. Such pseudo-progression might lead to the suspension of an active treatment.Few studies have addressed the potential of cfDNA testing for monitoring the response to immune-checkpoint inhibitors in patients with advanced melanoma. Two initial studies in small cohorts of patients receiving different immune-therapeutics (either PD-1 inhibitors or CTLA inhibitors or combinations) demonstrated a correlation between ctDNA dynamics and response to therapy ,65. In pBRAF, NRAS and cKIT most frequent variants, the criterion used by the authors was only whether the ctDNA was detectable or not. Interestingly, the response rate in patients with detectable ctDNA at baseline but undetectable after 12 weeks of therapy was similar to that in patients with undetectable ctDNA at baseline and after 12 weeks , and much higher as compared with that in patients with detectable ctDNA at baseline and after 12 weeks (6%). In addition, the first two groups had significantly longer PFS and OS as compared with the third group. The predictive value of ctDNA monitoring at 12 weeks was confirmed via multivariate analysis, thus suggesting that the clearance of ctDNA is a relevant predictive marker of response to immune-checkpoint inhibitors in metastatic melanoma. The same research group assessed the ability of cfDNA testing to identify pseudo-progression in a study including 125 metastatic melanoma patients who received anti-PD-1 antibodies alone or in combination with ipilimumab [The predictive value of ctDNA monitoring was next explored in a cohort of 76 patients with metastatic melanoma who received treatment with pembrolizumab or nivolumab monotherapy or in combination with ipilimumab . Althougilimumab . In thisilimumab .BRAF, NRAS and cKIT. By assessing a single mutation, the test might follow the dynamics of a single cell clone, which might not represent the behavior of the entire tumor in the case of tumor heterogeneity.Finally, some studies in small cohorts of patients tried to establish quantitative thresholds to define patients with response or progression at ctDNA analysis ,68. BothThe above-summarized data suggest that cfDNA testing might represent an adequate tool for monitoring responses to immune-checkpoint inhibitors in melanoma. However, prospective clinical trials are required to validate this approach in the clinic and to demonstrate that the early detection of tumor progression might allow a better therapeutic strategy.The use of NGS-based techniques might significantly increase the fraction of patients who can be monitored, improve the specificity and sensitivity of the test and provide information on tumor heterogeneity and the clonal evolution of the disease. For example, the NGS analysis of cfDNA from a patient with vaginal mucosa melanoma revealed the presence of two subclones with different responses to imatinib . One subBRAF and MEK inhibitors is associated with a high response rate in melanoma patients who carry a BRAF-V600 mutation [BRAF therapy in melanoma patients is due to the reactivation of the MAPK pathway by genetic or epigenetic mechanisms [PIK3CA signaling pathway.Combined targeted therapy with mutation . Howeverchanisms . HoweverBRAF and NRAS variants, NRAS mutations were detected in 3/7 melanoma patients progressing on targeted therapy with vemurafenib, dabrafenib or a dabrafenib/trametinib combination [NRAS mutations, in particular, at codon 61 (p.Q61K/R), in acquired resistance to dabrafenib/trametinib combination therapy in BRAF-mutant metastatic melanoma patients [NRAS mutations were detected in the cfDNA before the clinical and radiological progression of the disease.The feasibility of cfDNA testing in the assessment of acquired resistance to melanoma has been explored in a few studies. In a study that employed ddPCR to test bination . These dpatients . InteresNRAS and PIK3CA mutations not present prior to therapy were identified in melanoma patients progressing on targeted therapy with BRAF and/or MEK inhibitors [NRAS mutations were also found in the same cfDNA sample from a patient with progression, thus confirming the likely multi-clonal origin of acquired resistance to targeted therapies.By using the WES analysis of cfDNA, hibitors . MultiplNRAS were also identified in metastatic melanoma patients at progression following dabrafenib-and-trametinib treatment using the targeted sequencing of cfDNA [MAP2K1 and PTEN in a patient at progression, suggesting that these two genetic alterations might both contribute to resistance.Acquired mutations in of cfDNA . InteresBRAF gene amplification have been described as driving acquired resistance to BRAF/MEK inhibitors [BRAF gene amplification in 2/3 patients at progression [In addition to point mutations, other genomic alterations including hibitors . Indeed,gression .Evidence suggests that acquired resistance to targeted therapy is often polyclonal. In agreement with this hypothesis, distinct molecular alterations have been detected concurrently in the same tumor sample or among multiple tumor sites from the same melanoma patient progressing on targeted therapy . TherefoThe data summarized in this article clearly show that cfDNA analysis can offer important information on the prognosis of patients with melanoma in different stages of the disease. Furthermore, cfDNA analysis could allow a more accurate identification of patient candidates for targeted therapy or immunotherapy, through the representation of tumor heterogeneity and the early identification of patients who do not respond to therapies. However, the possibility of transferring this information into daily clinical practice depends on the resolution of a series of technological, biological and clinical issues.The introduction of NGS technologies for cfDNA analysis has increased the possibility of extending the test to a larger percentage of patients and has increased its clinical and analytical sensitivity. However, several NGS panels for cfDNA analysis are commercially available. For many of these panels, validation studies on adequate cohorts of biological samples are lacking, and external quality control programs are not yet available for these specific tests. Therefore, their introduction into clinical practice must be carried out with extreme caution, in order to avoid problems of false positives or negatives.BRAF mutations have not been associated with clonal hematopoiesis to date, the identification of other genetic alterations could still pose problems in the clinical interpretation of the data. At present, the contemporary analysis of DNA derived from leukocytes appears as the only approach to overcoming this limit.The use of NGS panels increases the sensitivity of the cfDNA test but also increases the possibility of identifying mutations associated with clonal hematopoiesis . This poThe analysis of cfDNA can provide information only on genetic alterations, which, alone, are not sufficient to represent the biological variability of neoplasms. In the era of precision medicine, the integration of the genomic profile with other biological omics, including transcriptomics, proteomics, metabolomics and epigenomics, that can be determined in biological fluids, represents a fundamental element for the advancement of knowledge on the pathogenesis and progression of cancer . A bioloThe various potential applications of liquid biopsies in the early diagnosis of recurrence and in monitoring the response to therapy must be validated in prospective clinical trials. In fact, it will be necessary to demonstrate that the early detection of disease recurrence allows the development of therapeutic strategies that result in a decrease in the mortality of patients with early stage melanoma. At the same time, it must be demonstrated that the modification of therapy in patients with metastatic melanoma who do not respond in terms of reduced ctDNA results in better survival.However, although there are many issues to be solved for the clinical implementation of cfDNA analysis, it can certainly be said that this technology opens up new scenarios in the management of patients with melanoma that could lead to important improvements in the diagnosis and therapy of this disease. In particular, the possibility of determining the overall profile of the genetic alterations of the neoplasm and of being able to evaluate its progression over time represents an important opportunity to improve the stratification of patients with melanoma for the purposes of therapeutic planning.


MAM locus is one of the most significant QTLs for glucosinolate diversity. However, most of what we understand about evolution at the locus is focused on only a few species and not within a phylogenetic context. In this study, we utilize a micro-synteny network and phylogenetic inference to investigate the origin and diversification of the MAM/IPMS gene family. We uncover unique MAM-like genes found at the orthologous locus in the Cleomaceae that shed light on the transition from IPMS to MAM. In the Brassicaceae, we identify six distinct MAM clades across Lineages I, II, and III. We characterize the evolutionary impact and consequences of local duplications, transpositions, whole genome duplications, and gene fusion events, generating several new hypothesizes on the function and diversity of the MAM locus.Glucosinolates are a diverse group of plant metabolites that characterize the order Brassicales. The  Glucosinolates (GSL) are a diverse class of amino-acid derived sulfur containing metabolites characteristic of plants of the order Brassicales . When thmethylthioalkylmalate (MAM) locus, also called the Elong locus, accounts for much of the natural variation observed in aliphatic GSLs  and to a lesser extent in Lineage II (Brassica and relatives). This work has been supported by large gene datasets, though with differing gene tree topologies  wherein they are reformed into a MAM1-like functional gene with partial MAM2 sequences, or vice versa  or based on A. lyrata MAM  .Naming conventions for nd MAMc) . The AraIPMS and MAM share a high sequence similarity and similar enzymatic function (PMS contains two conserved protein domains: a pyruvate carboxylase (HMGL-like), that is involved in the carbon condensation reaction, and a leucine allosteric domain (LeuA), that commits the protein to the leucine biosynthesis pathway forming a homodimer   . For eacIPMS/MAM genes using HMMER  and one encompassing the known IPMS gene clade (blue cluster). Of the syntenic clusters found in the MAM clade, the green cluster identifies the ancestral MAM position, what we will call the MAM-Ancestral locus, and is equivalent to the Elong locus. The orange cluster represents a transposed and retained MAM locus-specific to Lineage II of the Brassicaceae, which we will call the MAM-Transposed locus. The analysis also recovered the 4th cluster of an unnamed lineage of genes that have retained only a single HMGL-like domain and are found in both our outgroup and in-group genomes. The A. thaliana representative gene of this clade (AT2G26800) has been shown to play a role in seed amino acid concentration  event β, the hypothesized origin of MAM from IPMS  MAM genes experienced convergent evolution of their amino acid sequences, or (2) a gene fusion event of separate MAM types occurred sometime during the divergence of Lineage I MAM. The latter scenario is both the more parsimonious conclusion, and it is supported by the previous characterization of population-level gene fusion events at the Ancestral-MAM locus  and the rest of Lineage II  at each. Whereas in other genomes, like Sinapis alba, MAMa and MAMf genes remain paired although duplicated at separate loci. We propose that phenotypic differences between Brassica and Arabidopsis, such as the ability to co-synthesize different carbon chain majority phenotypes, are facilitated by the physical separation of MAM genes within the genome. By influencing the rate of diversification for MAM genes at the different MAM-Ancestral loci and allowing for novel genomic interactions, the WGT may have been a critical step in driving the specialized metabolic innovation we see in this dynamic crop lineage.Polyploidy offers another mechanism for  genomes . In BrasMAM/IPMS gene family serves as an excellent example of how a primary metabolic gene can, over millions of years and leveraging any source of novelty, give rise to a diverse lineage of highly adaptive specialized metabolic genes. Utilizing micro-synteny gene networks and broad phylogenetic sampling, we find that multiple modes of gene duplication have significantly influenced the evolutionary trajectory of the MAM locus and thereby diversity of aliphatic GSL profiles. By exploring some of the evolutionary consequences of whole-genome duplication, gene transposition, local duplication, and gene fusion, we have generated several new testable hypotheses as to the nature of MAM and GSL diversity. In the future, new experimental approaches and broad phylogenetically informed sampling will be critical to continue developing a robust understanding of this important gene family.The The datasets generated for this study can be found in the information provided in RSA performed research and wrote the manuscript. JCP helped design the study and edited the manuscript. MES designed the study and helped write and edit the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The establishment of the Age-Friendly University (AFU) network and adoption of the 10 principles by institutions of higher education, was a major advance in the United Nations Sustainable Development Goal of promoting healthy and active aging through opportunities for intergenerational communities. AGHE endorsed the principles in 2016, since then over 60 institutions have joined the global network. Tools are needed to identify benchmarks that institutions can use to assess progress toward realizing the AFU principles on their own campuses. This symposium shares work done at the University of Massachusetts Boston, to develop and refine the AFU Inventory and Campus Climate Surveys (ICCS), a survey-based assessment instrument (developed from a prior pilot study in 2018) based on the premise that it is necessary to assess both the institution’s actual age-friendly practices and its perceived age-friendliness or campus climate. In August, 2019, the University of Massachusetts President’s office endorsed the 10 principles for the entire UMass system of 5 campuses, presenting an opportunity to assess a multi-campus system. To date, we have surveyed UMass Boston, UMass Lowell, UMass Dartmouth and UMass Medical . Testing and refinement of the AFU ICCS will contribute to both short- and long-term recommendations to assist in strategic planning by higher education institutions. Whitbourne will present the Inventory reporting tool. Bowen will present the Climate Survey. Gautam and Revell will describe the AFU work at UMass Lowell and UMass Dartmouth respectively and the use of the assessment tools on their campuses; Morrow-Howell will serve as Discussant.


Polyoxometalates (POMs) has proved its advantage in constructing high-performance nanocomposite membranes such as catalytic membranes, adsorptive membranes, and forward osmosis membranes. However, it is challenging or tedious to characterize its distribution and effect on the membrane structures due to the equipment resolution limitation, discrete nano-scaled structures of POMs, and limited doping amount compared to the polymeric membrane matrix. In this paper, POMs-functionalized polyvinylidene fluoride (PVDF) membranes were fabricated by phase inversion combined with the sol-gel method, and electrochemical impedance spectroscopy (EIS) was utilized to analyze the nanocomposite membrane intrinsic properties. Through adjusting the additives in the sol-forming process, a set of membranes with varied intrinsic properties were developed accordingly. The wetting degree of the membranes related to the hydrophilic nature of the membrane surfaces had a crucial influence on the impedance measurements at the early stage. Through EIS analysis, it was demonstrated that the amination of the membrane matrix through (3-aminopropyl)trimethoxysilane (APTMS) treatment and the immobilization of POMs through electrostatic attraction would not generate new pore structures into the membrane and only alter the membrane surface roughness and composition. To my knowledge, it is the first time that EIS was utilized to characterize the hydrophilicity of the membranes and pore structures of the POMs-modified membranes. Our findings indicate that EIS can provide valuable information for probing the structures of other nano-materials-incorporated membranes. Membrane technology has been demonstrated to be efficient for water treatment over the past decades. Inspired by the multi-functional characteristics of biomimic membranes, incorporation of nanomaterials, namely nanocomposite membranes, has been a promising approach to tailor the membrane structure and impart additional unique properties in the past decade . Up to dPOMs are a unique class of metal–oxygen cluster species usually in nano-sizes ranging from ~5 Å to 40 Å, with versatile composition, scale, structure, charge distribution, and redox potential . They arElectrochemical impedance spectroscopy (EIS), as an economic and non-invasive characterization technique, can be potentially utilized to study the membrane structures ,17,18, m11O39Co(H2O)]4− (PWCo), was used as a model POM to fabricate the POMs modified membranes. It has proved as a potent catalyst in various reactions such as the removal of pesticides and dyes  (PWCo) was synthesized according to the literature . It. ItS forIt can be clearly seen that there was only one semicircle for each Nyquist plot and only one platform region in the Bode plots for all the membranes, suggesting a single time constant in the impedance spectrum. Therefore, though the two-step modification was applied, the membrane model still consists of two compartments: the membrane matrix and the empty volume of the pores. The introduction of APTMS and POMs would not induce the formation of any new kind of pores in the membrane, while the surface roughness and composition were changed. POMs-functionalized PVDF ultrafiltration membranes were fabricated in two routes involving the sol-gel method at different membrane preparation stages. Several conventional characterization techniques were performed to differentiate the membranes. The effects of POMs on the membrane properties were probed by EIS and analyzed based on the equivalent circuit. EIS results showed that the amination of the membrane matrix through APTMS treatment and the immobilization of POMs through electrostatic attraction did not generate new pore structures into the membrane, while the membrane surface roughness and composition were changed. The incorporation of POMs enhanced the membrane hydrophilicity without deteriorating the membrane pore structure and sacrificing the water permeation. It was proven that the wetting degree of the membrane relating to its hydrophilic nature had a crucial influence on the impedance measurements at the early stage. After associating EIS analysis with the evolution of immersion time, the hydrophilic property of the membranes along with their pore structures could be concluded. Therefore, EIS can be potentially applied for probing the structure evolution of different nano-materials-incorporated membranes.


Family caregivers of older adults report lack of preparation for their role, particularly upon acute hospital discharge following a medical event. Addressing the needs of family caregivers in the acute care setting prior to hospital discharge requires the identification of the caregiver, an assessment of caregiver preparedness, and a plan of care to address gaps in preparedness. The Preparedness Assessment for the Transition Home 7-item (PATH-7) is a valid and reliable instrument developed to assess family caregivers readiness for the caregiving role during acute care. The PATH-7 paper-pencil self-administered assessment was implemented in clinical care in medical-surgical nursing units in 2 acute care hospitals. Interventions to address gaps in preparedness were selected from a catalogue of interventions to develop a caregiver plan of care. The most frequent challenge identified by family caregivers was fulfilling the caregiving role on top of their other roles and responsibilities. This illustrated the need to assist family caregivers with exploring options for recruiting others to help with their roles and responsibilities and identify solutions soliciting and organizing help. This novel program promotes addressing the needs of the family unit, moving to a family-integrated are delivery model. Implementation challenges included in-person contact with caregiver to administer assessment, resources to respond to identified gaps in readiness, and lack of technology-enabled assessment administration. Positive staff experience with identifying and addressing needs of caregivers was a facilitator of staff engagement. Identifying, assessing, and addressing the needs of family caregivers of older adults is feasible in the acute care setting.


We review recent developments that suggest that autophagy plays a role in lipid homeostasis in plants in a manner that is comparable to algae, yeast, and animals. Autophagy is a universal mechanism that facilitates the degradation of unwanted cytoplasmic components in eukaryotic cells. In this review, we highlight recent developments in the investigation of the role of autophagy in lipid homeostasis in plants by comparison with algae, yeast, and animals. We consider the storage compartments that form the sources of lipids in plants, and the roles that autophagy plays in the synthesis of triacylglycerols and in the formation and maintenance of lipid droplets. We also consider the relationship between lipids and the biogenesis of autophagosomes, and the role of autophagy in the degradation of lipids in plants. Autophagy (‘self-eating’) is a universal mechanism that facilitates the degradation in eukaryotic cells of unwanted cytoplasmic components. Two distinct types, micro- and macro-autophagy, have been reported to play roles in metabolism and nutrient recycling in plants. Micro-autophagy is characterized by the invagination of the tonoplast to trap cytoplasmic material and create autophagic bodies within the vacuole. Macro-autophagy involves the formation in the cytosol of double-membrane vesicles, called autophagosomes, that sequester cytoplasmic components and carry them to the lytic vacuoles where they are released. Up to 40 autophagy genes (ATGs) are involved in the macro-autophagy machinery; 18 of them constitute the core machinery that participates in the formation of autophagosomes. By contrast with macro-autophagy, the molecular and genetic bases of micro-autophagy remain unclear. Links between macro- and micro-autophagy and plant primary and secondary metabolism have been documented in several reports. In this review, we highlight recent developments in the investigation of the role of autophagy in lipid homeostasis in plants by comparison with algae, yeast, and animals.As in other eukaryotic cells, lipids are found in membranes but are also stored as droplets in the plant cell. Triacylglycerols (TAGs) packed in lipid droplets (LDs) represent a major form of carbon storage. Oleaginous plants such as rapeseed, soybean, sunflower, and peanut accumulate numerous LDs in their seeds. Other plants such as avocado and olive store lipids in the pericarp of their fruits. Bio-membranes consist of double layers of polar lipids that are mainly composed of glycerolipids . Membranes also contain sterols and long-chain sphingolipids (LDs are formed of apolar lipids (i.e. TAGs and sterol esters) surrounded by a monolayer of phospholipids containing specific proteins. The LDs of the seeds are also referred to as oleosomes, as they are coated by a specific family of proteins called oleosins. The presence of oleosins at the surface of the seed LDs prevents the phospholipids from adjacent LDs coming into contact and fusing with them (de novo biosynthesis of fatty acids (FAs) takes place in the chloroplast, while TAG assembly occurs at the ER. Newly synthesized FAs are exported from the plastids and eventually modified by desaturation after incorporation into phosphatidylcholine (PC) in the ER. The assembly of FAs into TAGs occurs through different pathways that take place in the ER  and OLEOSIN-1, which enhance formation and accumulation of LDs, second with a mutation in trigalactosyldiacylglycerol1 (tgd1), which impairs the ER lipid pathway and enhances the chloroplast pathway for TAG synthesis, third with a mutation in SDP1, and fourth with a mutation in the plastid trigalactosyldiacylglycerol1 (act1), which controls chloroplast FA synthesis. However, using dark treatments, they showed that autophagy has no impact on the synthesis of FAs in the chloroplast and that the recycling of the thylakoid membranes is not modified in the autophagy mutants. Their electron micrographs suggested that the lipophagy of LDs occurs in a resembling micro-lipophagy that requires the macro-autophagy core machinery. atg5 and atg5/sid2 mutants with the Col-0 wild-type and the sid2 mutant to show that defects in autophagy triggered stress in the ER and increased the abundance of enzymes involved in lipid metabolism in the ER and peroxisomes, irrespective of nitrate or sulphate availability. Enzymes involved in the elongation of very-long-chain FAs together with the PDAT1protein were more abundant in atg5 and atg5/sid2 than in the control lines. Enzymes involved in the peroxisome β-oxidation pathway were also more abundant while chloroplast enzymes involved in FA synthesis were less abundant. In parallel with a decrease of chloroplast proteins in the atg5 mutants, a decrease of galactolipids was also observed. These decreases were more severe under conditions of nitrate and sulphate limitation than under control nutrition, showing that chloroplasts were degraded in the atg5 lines independently of autophagy when the plants were starved. Many proteases including SAG12 and AED1 are strongly increased in the atg5 mutant, suggesting that alternative pathways are involved in the degradation of chloroplasts when autophagy is defective  and sphingolipids  compared with control lines. This therefore suggests that the turnover of lipids originating from the ER and plasma membranes is impaired in autophagy mutants. The studies of Lipid degradation involving autophagy is referred as lipophagy, and lipid degradation by lipases in the cytosol is referred as lipolysis. Lipophagy can occur by both macro- and micro-autophagy. In algae and yeast both macro- and micro-lipophagy have been observed . This miThe role of autophagy in the mobilization of the TAGs stored in the LDs of seeds during the post-germinative phase has not yet been documented. The SDP1 lipase that is associated with the seed LDs is involved in the process of germination . It is lInterdependent relationships between autophagy and lipid homeostasis are supported by many studies in yeast, algae, and animals. Much less is known about the situation in plants, although some recent advances see  suggest Box 1.Disruption of basal macro-autophagy impedes lipid turnover and mobilization in the membranes of organelles, and the lipophagy that results in degradation of lipid droplets under carbon stress morphologically resembles yeast micro-autophagyatg5 mutants suggest a role of macro-autophagy in the turnover of the plasma membrane, and of the membranes of other organelles except for chloroplastsLipidomic and proteomic studies of Arabidopsis atg5 mutants exhibit an accumulation of phospholipids and sphingolipids and a decrease of galactolipids compared with the wild-type. This shows that the turnover of lipids originating from the ER and plasma membrane is impaired in the autophagy mutants, while chloroplast and thylakoid membranes may be degraded independently of autophagy.atg12 mutants suggest that they have modifications in lipid homeostasisLipidomic and proteomic studies of maize Multi-omic analyses performed by Degradation of ubiquitinated oleosins requires the PUX10-CDC48A protein and is a prerequisite to the degradation of lipid droplets in Arabidopsis seedsThe degradation of oleosins is a prerequisite for the post-germination mobilization of the lipids stored in seed lipid droplets (LDs).


Assisted reproductive technologies such as artificial insemination have delivered significant benefits for farm animal reproduction. However, as with humans, assisted reproduction in livestock requires the manipulation of the gametes and preimplantation embryo. The significance of this ‘periconception’ period is that it represents the transition from parental genome regulation to that of the newly formed embryo. Environmental perturbations during these early developmental stages can result in persistent changes in embryonic gene expression, fetal organ development and ultimately the long-term health of the offspring. While associations between maternal health and offspring wellbeing are well-defined, the significance of paternal health for the quality of his semen and the post-conception development of his offspring have largely been overlooked. Human and animal model studies have identified sperm epigenetic status  and seminal plasma-mediated maternal uterine immunological, inflammatory and vascular responses as the two central mechanisms capable of linking paternal health and post-fertilisation development. However, there is a significant knowledge gap about the father’s contribution to the long-term health of his offspring, especially with regard to farm animals. Such insights are essential to ensure the safety of widely used assisted reproductive practices and to gain better understanding of the role of paternal health for the well-being of his offspring. In this article, we will outline the impact of male health on semen quality , reproductive fitness and post-fertilisation offspring development and explore the mechanisms underlying the paternal programming of offspring health in farm animals. Such associations underlie the Developmental Origins of Health and Disease (DOHaD) hypothesis (in vitro and in vivo), affecting long-term offspring development and health (The development of efficient assisted reproductive technologies (ART) such as artificial insemination in cattle has increased the genetic gain in livestock dramatically . This hapothesis . Here, td health   has also been shown to affect the onset of puberty in bulls. Low nutritional intake in calves associated with increased pulsitility of luteinizing hormone and greater testicular development . InteresIn rodents and humans, similar effects on testicular function and sperm quality have been identified in response to a range of dietary and environmental factors such as over- or under-nutrition , endocriGNAS complex locus has been identified in sperm from males of lower fertility , epigenetics provides a molecular mechanism to better interpret the long-term effects the environment has on the emergence of certain phenotypes . Epigeneertility . Interesody mass  while getabolism . Similartabolism . The roltabolism . Sperm htabolism  and histtabolism  to the oand Sox2 . In cattd CRISP2 . Analysed CRISP2 . In addid CRISP2 . Furtherd CRISP2 .As with other aspects of semen quality, the age at which semen is collected from a bull may also affect the epigenetic status of the sperm. DNA methylation profiles have been shown to differ in sperm collected from early pubertal, late pubertal, and pubertal bulls . Separato C for 24 to 72 hours in commercial GEPS extender showed significantly higher motility, survival and integrity of the acrosome prior to preservation when compared to washed sperm held in GEPS alone (PTGS2) expression but also the expression of multiple maturation promotion factors within the oocyte, cumulus and granulosa cells within the ovary  in uterine endometrial tissue  have been identified in men with azoospermia . ConversIn addition to the protein composition of the seminal plasma, there is now a large interest in the role exosomes may have in regulating male fertility. Epididymal exosomes (epididymosomes) contain a range of proteins, microRNAs, tRNA-derived small RNAs (tsRNAs) and fluid and are able to interact with the mature sperm. Analysis of epididymosomes from mice have identified over 350 miRNAs with approximately 60% of them being detectable in the sperm . FurtherH19/IGF2 regions and the paternally expressed long non-coding RNA KCNQ1OT1  activity through both sperm and seminal plasma mediated pathways . Underlyin vivoenvironment, manipulation of the gametes and the use of sub-quality gametes. The latter can be a direct consequence of the health of the parents at the time of conception. Poor nutritional status in a range of animal models has been shown to negatively impact gamete quality, fertility and post-fertilisation development. While a significant focus has been to define and improve maternal reproductive fitness, sperm quality and an understanding of the potential long-term consequences of poor paternal health have remained overlooked. From a large animal model perspective, our understanding of the long-term paternal effects is still very limited. The majority of studies so far have been conducted in rodents and humans. While these have provided detailed mechanistic insight into the potential molecular and epigenetic mechanisms through which paternal health links to sperm quality and offspring well-being, data from mice and humans cannot always be extrapolated directly to other mammalian species. Furthermore, a greater understanding of the role of the whole semen, and not just the sperm in isolation, is needed to understand male reproductive fitness effects. In both agricultural and human ART, procedures are routinely conducted in a predominantly seminal plasma free environment followed by the transfer of an embryo into a non seminal-primed uterus. Data from rodent (As with human reproduction, ART in farm animals has revolutionised fertility management on a global scale. While the application of such technologies has enhanced our capacity to treat human infertility and make agricultural practices more efficient, we must be mindful of the potential implications for offspring health. Both human and animal ART are associated with significant implications for the resultant offspring. Here, both increases and decreases in fetal growth and weight at birth have been reported, resulting in complications with postnatal offspring wellbeing and development. Underlying such offspring effects are factors including a failure to recapitulate the natural maternal m rodent  and humam rodent  studies


The cornea is the most external layer of the eye and serves two important roles in (1) the refraction of light and (2) protection from the outside environment, both of which are highly dependent on the collagen assembly of the corneal stroma. This study sought to determine the collagen fiber arrangement of the canine corneal stroma and correlate the stromal organization with tissue stiffness in the anterior and posterior cornea. Collagen organization of the canine cornea was visualized through second-harmonic generation (SHG) imaging, and tissue stiffness of the anterior and posterior corneal stroma was determined by atomic force microscopy. Analysis of the canine anterior corneal stroma using SHG imaging documented intertwining of the collagen fibers with a high degree of fiber branching, with a more lamellar and non-branching posterior stroma. The anterior stroma had significantly higher tissue stiffness in both dogs and humans, when compared with the posterior corneal stroma . There was a direct correlation between corneal collagen stromal organization and tissue stiffness in the dog, which was consistent with other mammalian species previously examined and likely reflects the need for maintenance of rigidity and corneal curvature. The cornea is responsible for many functions of the eye, serving as the primary refractive interface for light entering the eye, as well as forming an anterior physical barrier to the external environment. The corneal stroma comprises nearly 90% of the total corneal thickness in most domesticated mammals, and is primarily composed of stromal collagen . The indSecond-harmonic generation (SHG) imaging is a nonlinear optical imaging technique that allows for visualization of collagen fiber arrangement . BrieflyThe organization of the collagen fibers of the corneal stroma has been shown to be directly correlated with corneal stromal stiffness. Using indentation techniques, such as atomic force microscopy (AFM)-based nanoindentation, it is possible to assess the stiffness of unfixed biologic tissue. In both the human and rabbit cornea, the anterior stroma has been shown to be stiffer than the posterior stroma, likely due to the differences noted in the degree of collagen fiber intertwining between the anterior and posterior stroma ,8. The cThe purpose of this study was to characterize the collagen fiber organization in the canine cornea utilizing SHG imaging and determine if stromal arrangement correlated with the tissue stiffness of the anterior and posterior corneal stroma. We hypothesized that the collagen fiber arrangement within the canine cornea would have similarities to other previously characterized mammals, and that the corneal stromal stiffness would differ based on the collagen fiber organization between the anterior and posterior stroma. Canine globes  from seven-month-old purpose bred Beagles were procured from animals that were euthanized for reasons unrelated to the current study. Immediately after euthanasia, the eyes were enucleated and transported overnight chilled and wrapped in gauze soaked with phosphate buffer solution (PBS) for processing the following day for either SHG imaging or AFM. On examination, the globes were deemed free of corneal disease.Human corneal buttons (n = 2) were procured from Saving Sight  stored in Optisol  at 4 °C and used at less than or at 3 weeks postmortem. The selected human corneal buttons lacked historical or observable corneal stromal pathology.A single canine globe was fixed by perfusion under pressure using 4% paraformaldehyde in PBS (pH 7.4). The cornea was then dissected from the remainder of the globe and bisected mid-sagittally. One of the sections was vertically embedded in low melting point agarose  and a vibratome  was used to cut multiple 300 μm thick sections. The cut sections were then stored in PBS until imaging. Cross-sectional imaging of the cornea was performed as previously described ,8. BriefFor the canine globes, an 8 mm diameter corneal trephine was used to mark the central cornea and the corneal epithelium was removed using an excimer laser spatula  within the marked region. An excimer laser  was used to photoablate the epithelial basement membrane and stroma to expose either the very anterior corneal stroma (1 cycle of 6 mm diameter at 40 Hz with 83 pulses ablated at 50 μm depth) or the posterior corneal stroma  in the center of the epithelial wound. The cornea was then carefully dissected from the remainder of the globe. Human corneal buttons that are unsuitable for transplant generally have fewer corneal endothelial cells resulting in edema. Thus, the excimer laser treatment depth was adjusted to factor for edema while attaining appropriate stromal depth. Subsequently, for the human corneal buttons, the corneal epithelium was removed using an excimer spatula and an excimer laser was used to photoablate the epithelial basement membrane and stroma to expose the anterior corneal stroma  or the posterior corneal stroma , and the positioning within the stroma was confirmed using spectral domain optical coherence tomography  .Using the Soft-Clamping Immobilizing Retainer of Tissue (SCIRT) method, the cornea was placed into an AFM dish that had been coated with cured dielectric silicone  with the anterior-most aspect of the tissue oriented upwards . The SCIThe D’Agostino-Pearson test was performed to assess the normality of the data, then a two-tailed Mann-Whitney ranked sum test was performed on the AFM measurements from the anterior and posterior corneal stroma using Prism 8 . Data was represented as box and whisker plots with the box extending to the 25th and 75th percentiles, with the median as the line in the middle of the box and whiskers representing the minimum and maximum values. In order to compare the elastic moduli obtained from canine corneas with those from human and rabbit tissues, the mean modulus measurements and standard deviation were calculated.Using SHG imaging, the orientation and alignment of the corneal collagen fibers were visualized with the fixed specimen from limbus to limbus . The antp < 0.0001; p < 0.0001; The median tissue stiffness of the canine anterior stroma (1.3 kPa) was significantly increased (nearly 4-fold) when compared with the posterior stroma (0.3 kPa)  . InteresThe consistent finding of a high degree of intertwining in the anterior corneal stroma amongst mammals suggests that there is a significant evolutionary pressure to maintain this characteristic of the cornea for rigidity, curvature and light refraction. The relationship between the three-dimensional organization of the collagen fibers and the biophysical attributes of the cornea suggests that the mechanobiology of the cornea would be impacted by certain corneal pathologies and surgical interventions. For example, canine patients that undergo superficial keratectomy for superficial corneal pathology or spontaneous chronic corneal epithelial defects (SCCEDs) would result in extirpation of this important area of intertwined collagen fibers. These animals would likely have permanent alteration in the rigidity of the anterior stroma. This is in addition to induced altered corneal curvature, light refraction and vision, features essential for service animals and others requiring high visual acuity.There is a clinical need to develop synthetic and bioartificial corneas for use in transplantation in patients with corneal pathology; therefore, the development of models to study transplant physiology and serve as a source of xenograft material is critically important. However, if there are key biomechanical differences between seemingly identical tissue at the light microscopy level, these differences may represent significant complications in the utilization of xenograft material. The data presented in this study demonstrate that, despite the overall similarity in collagen arrangement between humans and dogs, the different percentage of collagen fiber intertwining in the cornea leads to important variations in tissue stiffness. To more thoroughly investigate the structural differences that lead to variations in tissue biomechanics, further investigations are required that focus on (1) quantification of collagen fiber intertwining; (2) definition of glycosaminoglycans and proteoglycans composition that bridge elastic and collagen fibers; (3) determination of corneal ultrastructure with serial block face scanning transmission electron microscopy (EM) or cryopreserved EM; and (4) the organization of the stromal matrix using x-ray scattering or multi-photon techniques. When viewed from the clinical application of transplantation, the reduced tissue stiffness in the anterior stroma of non-human mammals may be a significant impediment to transplantation into humans due to alterations in corneal curvature and refraction. Also, species-based considerations may be essential when evaluating the safety and efficacy of responses to drugs for treating corneal disorders. Therefore, the design and generation of biosynthetic and bioartificial corneal tissue will likely be highly influenced by the collagen organization and biomechanical attributes.The values obtained for tissue elastic modulus of a biologic tissue can vary dramatically depending on the specific method used (tensile vs. indention) . Even whOverall, this study determined that the organization of the canine corneal stroma is similar to other mammalian species and is characterized by the presence of intertwining of the collagen in the anterior stroma. Tissue stiffness directly correlated with the degree of intertwining of the stromal fibers, with the anterior stroma being stiffer. These studies provide a touchstone for future studies evaluating the consequence of corneal pathologies and surgical interventions on the mechanobiology of the cornea.


Potentially inappropriate medication (PIM) use has adverse effects on health, particularly in elder patients. Various country-specific explicit criteria have been developed to measure the appropriateness of prescribing worldwide. However, it is difficult to apply the criteria developed from other regions to measure and guide the local prescribing practice in Hong Kong. This study aims to develop a Hong Kong-specific PIM assessing tool from previously published criteria and validate this tool using the modified Delphi method.A disease-oriented Hong Kong-specific preliminary PIM list was developed based on nine sets of reference criteria selected from a literature review. Any medication or medication class appeared in at least two sets of the reference criteria as well as its related medical conditions were selected as PIM candidates. After examining the availability of PIM candidates by the Hong Kong Hospital Authority drug formulary, the Hong Kong-specific preliminary PIM list was validated by a two-round of modified Delphi process. Eight experts from different specialties were invited to rate the degree of inappropriateness of each PIM candidate using a five-point Likert scale. The experts were also encouraged to propose therapeutic alternatives and new PIM candidates not covered by the preliminary PIM list. The PIM candidates that the expert panel didn’t reach consensus on were excluded from the final Hong Kong-specific PIM list.After two rounds of the Delphi process, eight PIM candidates remained questionable and thus were excluded from the PIM list. The final Hong Kong-specific PIM list included a total of 164 statements applicable to older adults aged 65 years or above, among which 77 were under PIMs independent of diagnoses, and 87 were under PIMs considering specific medical conditions.The Hong Kong-specific PIM list can be used as a quality measure and an educational tool to improve the local prescribing quality. Further studies should validate its association with adverse health outcomes in clinical and research settings.The online version contains supplementary material available at 10.1186/s12877-021-02024-0. Potentially inappropriate medication (PIM) use, generally occurring when patients are prescribed drugs with an unfavorable balance of benefits and risks, is a preventable problem . PIM useWith an aging population, appropriateness of prescribing has become a public health concern worldwide and thus, various tools have been developed to assess the quality of prescribing. These measures can be generally categorized into explicit (criterion-based) and implicit (judgment-based) tools . ExpliciOlder adults are usually excluded from clinical trials, thus lacking evidence on balancing risks and benefits of therapeutic agents in this population . Under tIn Hong Kong, we recently conducted a study to assess the prevalence of PIM use in elder patients aged 65 years and older visiting general outpatient clinics, using the main subsets of the 2015 Beers criteria. The prevalence estimates ranged from 55.6% in 2006 to 47.5% in 2014, which were relatively higher than those reported in western countries , rangingThe first step in developing the Hong Kong-specific PIM criteria was to establish a preliminary drug list based on existing criteria in the literature. A literature search was conducted to identify explicit tools assessing PIM use on the PubMed database from January 1991 to April 2019 internationally and consequently, 40 sets of validated explicit criteria were identified. Explicit criteria that 1) applied to the general population aged 65 years and older, 2) used the literature review on safety and efficacy of drug use as an evidence base, and 3) described the methods of development and validation for the criteria, were taken into account. Regarding the criteria that were updated occasionally, only the latest versions of PIM lists were considered and the early versions of the criteria were eliminated from the study. Consequently, the following nine sets of explicit criteria were included as reference criteria in the current study: the McLeod criteria , the RanThe structures of the nine sets of reference criteria were heterogeneous, with the classification system of criteria statements as disease-oriented or/and drug-oriented. The evaluated aspects of the reference criteria are summarized in Table Based on the structure of the preliminary PIM list, the operational definitions of PIM use in the current study are described as follows: 1) medications or medication classes that should be generally avoided independent of diagnosis or conditions and 2) medications or medication classes that should not be used in older adults known to have specific medical conditions. Other evaluated aspects of PIM use including dosage, duration of therapy, alternative therapies and special considerations of use were supplemented by experts’ comments during the Delphi process. Following the operational definition of PIM use in this study, seven sets of reference criteria included generally avoided drugs independent of diagnoses and six sets of criteria included disease-drug interactions. After extracting all related statements from the reference criteria, 279 medications/classes under generally avoided drugs independent of diagnoses and 115 medications/classes considering specific medical conditions were identified.To prepare a manageable amount of PIM statements for further evaluations in the Delphi process, any medication or medication class appeared in at least two sets of the reference criteria  and its related medical conditions were selected from relevant reference criteria as candidate PIMs for each category of PIM use. We used the Beers criteria to select medications that appeared in two sets of criteria because the Beers criteria were considered as the golden standard for PIM assessment and many country-specific explicit criteria were developed based on the Beers criteria . The avaThe preliminary PIM list was validated by a two-round modified Delphi process. Experts from different specialties were recruited by invitation emails and personal communication, all involved with practice or research in medication issues for elder patients for at least five years. A face-to-face meeting was also convened with six potential experts one by one to brief them on the study background and objectives. After the briefing meeting, five experts decided to participate in the project. Invitation emails were also disseminated to the Hong Kong Geriatric Society (HKGS). Four geriatric physicians from the HKGS replied for a further in-person meeting and three of them finally decided to take part in the study after the meeting. Overall, a total of eight (80%) experts agreed in participating in the project seeking to develop the Hong Kong-specific PIM list. On the basis of the preliminary PIM list, a five-point Likert scale was designed to construct the questionnaire for collecting experts’ ratings and comments during the Delphi process. The questionnaire was examined for wording and content validity by three qualified experts.The first round of the Delphi process began in August, 2019. The experts were asked to rate the degree of inappropriateness of each candidate statement from a score of 1 (strongly disagree) to 5 (strongly agree). A score of 3 was neutral or undecided. Furthermore, the experts were encouraged to provide comments on any exception or restriction in terms of indication, dose, route of administration, or therapy duration to make each candidate statement more accurate. They were also asked to propose therapeutic alternatives to the PIMs independent of diagnoses and suggest new PIM candidates not included in the questionnaire.After the first round of questioning, the experts’ answers were presented as median with interquartile ranges (IQR) for each candidate PIM. The statements with medians ≥3.5 and IQR ≤ 1.5 were included in PIM use, whereas the statements with medians < 3.0 were excluded from PIM use . The staEight experts participated in the first round of the Delphi process. One expert with a pharmacy background quit during the second round of questioning due to illness. The characteristics of eight experts are shown in Table In the first round of the Delphi process, 117 (69.6%) candidate statements were designated as PIM use whereas 8 (4.8%) statements were designated as non-PIM use and dropped from the study. Forty-three (25.6%) candidates were classified as questionable PIM use, which required further evaluations in the second round. Furthermore, three new indications of the PIMs listed in the preliminary list were suggested by the expert panel to be included in the second round of rating. According to the ATC classification system, terazosin is classified as a urological drug while prazosin and doxazosin are classified as antihypertensives; however, they all belong to alpha-adrenoreceptor antagonists and can be indicated as either a urological or an antihypertensive drug based on the expert panel’s comments. The indications not included in the first round of questioning were rated again in the second round. The expert panel also proposed nine new PIM candidates not incorporated in the preliminary list, seven of which were under PIMs independent of diagnoses, and two were under PIMs considering specific medical conditions. Furthermore, the experts’ comments in terms of rationales, restrictions, and exceptions for each PIM candidate were consolidated and expanded based on the reference criteria. Consequently, a total of 55 candidates, including 44 in the current list plus 12 new suggestions, was brought forward to the next round of the Delphi voting.In the second round of questioning, 47 85.5%) candidates were rated as PIM use while 8 14.5%) candidates remained questionable PIM use Table . Among t4.5% cand.5% candiThis study developed and validated the first explicit tool for assessing PIM use in older adults in Hong Kong. Many country-specific explicit tools have been developed by including all the PIMs defined by several previously published criteria and validated in the local context –31. HoweAmong the 164 statements classified as PIM use in the Hong Kong-specific PIM list, experts had agreed with approximately 70% of them after the first round of questioning. This result may indicate that strong evidence could prove the pharmacological inappropriateness of these statements in older adults or better therapeutic alternatives exist in the public sector in Hong Kong. By contrast, some of the statements were not designated as PIM use until the second round of rating, such as certain hypnotics and sedatives (zopiclone and zolpidem), which may be because the expert panel believed that these drugs could be used with low dose or in short term depending on individualized situations in the context of Hong Kong. The proton pump inhibitors (PPIs) were designated as non-PIM use after the first round of questioning in this study. It is probably because the experts think PPIs can be used with caution among elder patients as long as the therapy duration is not longer than 8 weeks. Another reason could be the experts believe the misuse of PPIs is not particularly problematic for older adults, while the development of the Hong Kong-specific PIM criteria is unique to the aging population.After two rounds of the Delphi process, eight PIM candidates still remained questionable PIMs. Several reasons accounted for the questionable PIMs that the panel didn’t reach consensus on. First, some of the PIM candidates were regarded as inappropriate use in older adults in the reference criteria but no therapeutic alternatives existed in the HA drug formulary (e.g. fluoxetine as an antidepressant). Second, the expert panel decided that some of the questionable PIMs were acceptable for use in older adults as long as the dosage was not excessive . Third, several disease-drug interaction candidates were rated as undecided or neutral because the expert panel felt it was necessary to balance the benefits and risks of the potentially inappropriate drugs depending on the individuals’ circumstances. According to the previous studies conducted in other countries , 14, queApart from the preliminary PIM list, the panel also proposed nine new PIM candidates, seven of which were eventually designated as PIM use. However, some of these newly suggested statements were only applicable to a subgroup of older adults, such as bisphosphonates to bedbound patients, and lacked of evidence from reference criteria. Therefore, more research findings are necessary to justify the inappropriateness of these seven PIM statements in the future updates.Compared with the Beers criteria, the Hong Kong-specific PIM criteria are more user-friendly to guide the local prescribing practice. All the individual medications that appeared in the Hong Kong-specific criteria were listed with ATC classification codes, making it easier for health professionals to identify active substances. Therapeutic alternatives to PIMs independent of diagnoses are also offered to health professionals for reference. To be externally valid, explicit indicators for PIM use with adequate validity should be linked with adverse health outcomes. Some previous studies showed a positive association between the Beers criteria and multiple patient-related outcomes including hospitalization, mortality, falls and functional decline . The relThe Hong Kong-specific PIM criteria can be used to assess prescribing patterns in Hong Kong and to educate patients and health professionals on appropriate drug use. The statements listed in the Hong Kong-specific PIM criteria should be generally avoided in older adults aged 65 years or above from a population level. However, the criteria cannot replace a clinician’s final decision-making from an individual level. If patients are not responsive to the therapeutic alternatives, the potentially inappropriate medications included in the criteria could also be prescribed for elder patients with close monitoring and dose adjustments under specific circumstances. The development of the Hong Kong-specific PIM criteria is intended to raise awareness of difficulties of pharmacotherapy for elder patients. Because all the medications included in the Hong Kong-specific PIM list are covered by the HA drug formulary, they can be integrated easily into the HA computer system and updated on a cycle.The limitation of the Hong Kong-specific PIM criteria was that the preliminary PIM list was derived from previously published criteria with insufficient evidence on drug use from recent findings. The current study was intended to develop a disease-oriented PIM list and only two categories of PIM criteria were considered. Thus, expanding the Hong Kong-specific PIM criteria and including other categories of PIM criteria such as drug-drug interactions or duplications in future updates is necessary. Furthermore, the expert panel consisted mainly of physicians and pharmacists from public hospitals and the newly developed criteria were only applicable in the public sector. Thus, to facilitate the effects of the Hong Kong-specific PIM list, the selection of PIM candidates and the expert panel from the private sector should also be considered in the development of future versions of PIM lists.In conclusion, the Hong Kong-specific PIM list can be applied as a quality measure and an educational tool to improve the appropriateness of prescribing in the local context. It can provide physicians and pharmacists with practical advice to make better therapeutic decisions. Future studies should focus on validating the association between the Hong Kong-specific PIM assessment tool and adverse health outcomes in clinical and research settings.Additional file 1:. The full list of medications or medication classes with ATC codes included in PIMs considering specific medical conditionsAdditional file 2:. Main concerns and possible therapeutic alternatives of the Hong Kong-specific potentially inappropriate medication list


This commentary, in particular, addresses the challenges associated with the use of miRNAs and the advantages of nonviral methods of delivery in respiratory diseases.Chronic respiratory disorders are among the most common causes of severe illness and death worldwide. Globally, approximately around 4 million people die annually prematurely due to chronic respiratory diseases . miRNAs in vivo [Several biological and physicochemical factors are associated that minimize the delivery of miRNA to the target cells. These include low plasma half-life: in addition to a lack of stability in biological systems and fluids, primarily blood, the short-lived nature of their gene silencing effects is one of the principal obstacles in the use of miRNA. These are enzymatically degraded by cellular nucleases and quickly removed through the kidneys due to their low molecular weights. Studies show that naked miRNA quickly degrades in plasma having half-life of seconds to 30 min in vivo . Immunotin vivo . Secretiin vivo . Off-tarin vivo .miRNA inhibitors/antagomirs (AMO) are synthetic miRNA antagonists, also known as miRNA silent agents. They are commonly found bound to the miRNA. They mask the target site, which essentially avoids interaction with the miRNA, enabling them to be translated. This approach has the benefit of annulling the likely off-target effects of a wide range of miRNAs . Like AMNanotechnology has made significant strides in recent years, in both developing new materials and also with their applications. These advancements have led to the development of new DNA and RNA delivery systems to monitor diseases that can be used, instead of viral vectors . InorganIRAK1 target gene expression to 40% [et al. prepared nanoparticles of miR-126 with cationic polymers and demonstrated their ability to promote the incorporation of miRNA into the CFBE41o cells , thus significantly decreasing the expression of TOM1 [et al. developed a codelivery system that comprised of solid lipid nanoparticles enclosing miRNA-34a and paclitaxel (PTX) for synergistic effect to target lung cancer cells. The drugs administered co-operatively and more effectively blocked the metastatic production of BF10-CD44+ originating in the lung. In addition, this approach also decreased the doses of PTX, thereby, reducing the side effects associated with PTX alone [et al. in 2020, which also encapsulated myricetin and siRNA in mesoporous silica nanoparticles and showed its synergistic effect to suppress tumor growth in lung cancer cells with lesser side effects [One of the recent attempts made was to deliver miR146a using polymeric nanoparticles for the treatment of chronic obstructive pulmonary disease. Results showed that miR-146a has maintained its gene and protein functional structure. The high concentration of miR-146a-nanoparticles decreased the n to 40% . McKiern of TOM1 . Neutral of TOM1 . When co of TOM1 . Shi et TX alone . A similThe expanded and in-depth research in understanding the roles of miRNA opens up new possibilities for pulmonary disorders. The blend of miRNA with nanotechnology paves the way to achieve enhanced cellular uptake, endosomal escape and improved bioavailability. However, there are several challenges related with the unknown structure and stoichiometry of polymers along with the accumulation of nanoparticles in normal organs. Other key issues awaiting further exploration include effective gene silencing effect with absolute safety and the challenge to increase the delivery of the target substances to the organ of interest or even the cell of interest alone.