Anatomically modern humans originated in Africa and separated from their most likely recent common ancestor hundreds and thousands of years ago . They foThere is increasing evidence to suggest that the differences in the susceptibility of many common and rare diseases are primarily attributed to human populations’ diverse cultural, environmental, demographic, and genetic histories. The comprehensive understanding of fine-scale population evolutionary history will gradually change our understanding of the genetic architecture of diseases . Thus, tvia low-density genetic markers. It is also now possible to capture and sequence ancient DNA from ancient samples, which could provide pivotal insights into the formation of spatiotemporally diverse populations with unprecedented resolution. Although huge nationwide biobanks for characterizing the genotypes and phenotypes of millions of people have been established  . FIGG, oForensic investigative genetic genealogy and fine-scale structure of Human Populations”. In detail, this Research Topic aimed to characterize the genetic background and demographic history of ethnolinguistically and geographically diverse populations based on different densities of genetic marker. It would be helpful in exploring the long-range familial searches and fine-scale genetic localization within subgroups in other continents. This Research Topic attracted research focused on the basic knowledge exploration of the genetic background of one targeted population and included applied research focused on developing and validating forensic amplification systems.To summarize the new advances in FIGG and fine-scale population structure and illuminate the importance of full-scale genetic structure and diversity as the basis for the FIGG, we organized this Research Topic of the “Zhou et al. generated and analyzed genome-wide data of Liaoning Han people and found that genetic differences existed in geographically different Sinitic-speaking Han populations, which might result from other migration and admixture events of Hans during the period of “Chuang Guandong”. Hou et al. investigated the population history of Liaoning Mongolians based on ∼700,000 SNPs and provided new insights into the admixture history of Mongolic-speaking Mongolians according to shared allele-based analyses. He et al. explored the demographic history of Qiang people based on Eurasian modern and ancient reference populations. This study revealed that the Tibeto-Burman-speaking Qiang people derived their primary ancestry from Tibetan-related ancestral populations in North China. Wang et al. performed a genome-wide association study on 26,806 Chinese individuals. They identified 21 SNPs associated with widow’s peak, unibrow, double eyelid, earlobe attachment, and freckles. This study may facilitate a better understanding of the genetic basis of facial development in Chinese populations and provide new markers for forensic phenotype predictions. These studied Han, Mongolian and Qiang people are widely distributed in North China, and other populations from southern China and surrounding regions need to be further explored based on the array or sequencing data, such as Austronesian, Austroasiatic, Hmong-Mien and Tai-Kadai people. Generally, population genetic studies showed a strong correlation between Chinese cultural language and geographic patterns and population structure.Recent studies have demonstrated that population history reconstruction leveraging high-density genetic markers could uncover previously unrecognized population structures at a fine scale compared with forensically relevant loci . Zhou etGorin et al. selected 5,229 AISNPs and tested various mathematical models for biogeographic ancestry inference. The results showed that the accuracy of the prediction of this panel on one of 29 studied ethnic groups reached 71% and the proposed method could be employed to predict ancestries from Russian and neighboring populations. Huang et al. developed a machine learning approach for estimating the relationships with high error SNP profiles and found that this approach was more accurate and robust than the individual measures.The estimated patterns of genetic diversity in China can help accurately biogeographic ancestry inference and FIGG. Similar population stratifications were also identified in Siberian populations. Currently, available tools could effectively distinguish populations with different continental origins, but most of these are not efficient for differentiating people within the same continent. Huang et al. validated the forensic performance of a novel multiplex autosomal STR panel (including six CODIS STRs and 20 non-CODIS STRs). They found that this novel kit could be applied as a promising tool for forensic human identification and complex paternity analysis. Than et al. genotyped seven Lao Isan and three Laotian populations using Verifiler plus PCR Amplification kit. The allelic frequency results provided the genetic background of Austroasiatic and Tai-Kadai people from Laos and Thailand.STR genotyping has been applied in forensic investigations for nearly 30 years . NowadayLiu et al. developed and validated a six-color fluorescence multiplex panel including 59 autosomal InDels. Subsequently, Tibetan groups from China have been genotyped using the newly-developed 59-plex InDel panel. The comprehensive population genetic analyses showed that this homemade panel could be used as a powerful tool for individual forensic identification and paternity testing in Chinese Tibetan groups. Jin et al. developed a Next-Generation Sequencing (NGS) InDel panel, including 17 multi-InDels on the X chromosome. They found that the newly-developed panel could be adopted as an effective tool for individual forensic identification, paternity testing, and biogeographical ancestry inference.InDel loci, the second most abundant polymorphism across the human genome, possess low mutation rates and small amplicon lengths compared with STRs , which hJia et al. sequenced complete mitochondrial genomes of 146 Daur individuals in China. The results showed that the Daur ethnic group has high maternal genetic diversity and may have experienced recent population expansion. He et al. developed and validated the AGCU-Y30 Y-STR panel and conducted a Y-STR-based study to explore the paternal history of the Qiang people. The validated results showed that the novel Y-STR kit was sensitive and robust enough for forensic applications. Population genetic analyses revealed that the Qiang people are closely related to lowland Tibetan-Yi corridor populations.Genetic surveys based on uniparentally inherited markers have identified many paternal and maternal founding lineages in regional populations and their corresponding expansion events . Jia et WCH and ChinaMap, were recently launched to explore the genetic features of under-represented populations. Characterizing the genetic architecture of ethnolinguistically and geographically diverse populations will promote our understanding of population origin, separation, admixture, adaptation, and gene flow from archaic individuals.Human genetics needed the fundamental foundation supporting large-scale population genomic projects to characterize the full landscape of human genetic diversity and population structure, such as NHLBI TOPMed , gnomAD Knowledge of the fine-scale genetic structure and population-specific genomic database is the base for FIGG. Synthesizing our growing knowledge of evolutionary history with forensic investigations will help to achieve the promise of long-range familial searches and fine-scale genetic localization. For human population genetics and FIGG, we also had some challenges that needed to be overcome in the next step:Firstly, European bias in worldwide human genetic studies and Han Chinese bias in Chinese cohort research hinder in heath equality of genetic studies and forensic genetics. More population genomic studies from under-reported populations need to be conducted to characterize their uncharacterized genetic polymorphisms and the genetic spectrum of different genetic markers.Second, a population-specific genomic database should be constructed to provide a comprehensive landscape of different genetic markers and even include structural variations and mobile elements via the PacBio sequencing plateau.Third, cooperation of national genomic studies should be formed to promote data sharing in different institutions.Forth, population genomic projects should be conducted among included subjects with deep phenotypes, which would provide values for exploring the genetic basis for physical traits and medical phenotypes.Fifth, regional population-specific genomic datasets, FIGG panels, algorithms, and forensic databases should be developed and validated. FIGG has promoted successful inspection of criminal suspects among American or European populations, as there are publicly available European/American genomic databases and DNA. Land and GEDmatch severs. Similar databases from other countries or regions should be constructed in the future.Seventh, IBD-based algorithms need the high-density phased SNPs or whole genome sequencing data. Many forensic samples were highly degraded or had minute amounts of genetic materials. Thus, the primary focus is to develop more FIGG panels based on low-density SNP markers and non-linkage algorithms .In short, more ethnolinguistically and geographically diverse populations are needed to be studied based on different genetic markers and algorithm models, and population-specific panels based on different genetic variations and corresponding forensic databases need to be developed to achieve long-range familial searches and fine-scale genetic structure reconstruction.


A primary brain tumor glioblastoma is the most lethal of all cancers and remains an extremely challenging disease. Apparent oncogenic signaling in glioblastoma is genetically complex and raised at any stage of the disease’s progression. Many clinical trials have shown that anticancer drugs for any specific oncogene aberrantly expressed in glioblastoma show very limited activity. Recent discoveries have highlighted that alterations in tumor metabolism also contribute to disease progression and resistance to current therapeutics for glioblastoma, implicating an alternative avenue to improve outcomes in glioblastoma patients. The roles of glucose, glutamine and tryptophan metabolism in glioblastoma pathogenesis have previously been described. This article provides an overview of the metabolic network and regulatory changes associated with lipid droplets that suppress ferroptosis. Ferroptosis is a newly discovered type of nonapoptotic programmed cell death induced by excessive lipid peroxidation. Although few studies have focused on potential correlations between tumor progression and lipid droplet abundance, there has recently been increasing interest in identifying key players in lipid droplet biology that suppress ferroptosis and whether these dependencies can be effectively exploited in cancer treatment. This article discusses how lipid droplet metabolism, including lipid synthesis, storage, and use modulates ferroptosis sensitivity or tolerance in different cancer models, focusing on glioblastoma. Glioblastoma, designated as WHO grade 4, is the most common and most aggressive intra-axial brain tumor and has limited treatment options –3. The d; however, relapse remains inevitable. Temozolomide tetrazine-8-carboxamide  has sevimidazo5,-d (Lipogenesis (lipid acquisition) is an energy-expensive process of fatty acid synthesis and subsequent esterification to neutral lipids into lipid droplets. The primary process of synthesizing fatty acids from acetyl‐CoA subunits that are produced most commonly from carbohydrate catabolism is called is (DNL) . Acetyl-Glutamine is another non-lipid metabolite that contributes carbon to lipogenic acetyl-CoA Figure 2Acetate, often dispensable for cells, has also been shown as a source of acetyl-CoA for DNL in cancer cells, especially primary glioblastomas and other cancer metastasis to brain , 74. In Cytoplasmic acetyl-CoA is then carboxylated by acetyl-CoA carboxylase (ACC) to produce malonyl-CoA , 60, 65,De novo synthesized fatty acids are then further esterified to neutral lipids and stored in their nontoxic  form in lipid droplets , the indication of its level during Western diet consumption  warrantsr growth , 82, 83.de novo synthesis of fatty acids can be extended to the formation of either saturated or unsaturated fatty acids. Unsaturated fatty acids can be further classified into monounsaturated fatty acids  and polyunsaturated fatty acids . Unsaturated fatty acids can bind membrane phospholipids in differential degrees that subject tumor cells to oxidative stress. Studies have revealed that an increase in the MUFA/PUFA ratio results in fewer peroxidation-susceptible targets, ultimately reducing the susceptibility of cancer cells to ferroptosis (de novo synthesis) of these PUFA is necessary for producing ferroptosis death signals . Using genetic ablation and pharmaceutical inhibition, it has been shown that ACSL4 is a rate-limiting enzyme to catalyze the conversion of long-chain PUFAs, such as arachidonic acid and eicosapentaenoic acid to PUFA-CoA, and ultimately this increases lipid peroxidation and decreases resistance to ferroptosis –88. Undein vivo, DGAT inhibition induces significant cytotoxic effects, especially when combined with dietary long-chain PUFAs (LC-PUFAs), implicating the combination of diet (LC-PUFAs) and DGAT inhibitor (DFATi) administration as a highly relevant therapeutic combination to induce ferroptosis autophagy. Lipophagy is a process that tags specific lipid droplets and traffic whole lipid droplets to lysosomes for bulk degradation  and autophagy-associated gene 5 (ATG5), a cargo receptor of lipid droplets, can prevent induced lipid peroxidation and subsequent ferroptosis , 106. MeAlthough emerging evidence reinforces the idea that the amount of lipid and the localization of lipids in lipid droplets potentially affect the ability to induce ferroptosis in cancer cells, studies on the effect of lipolysis and lipophagy on sensitizing glioblastoma cells for ferroptosis are limited. There is limited information on how microenvironment and disease progression affect the biogenesis and breakdown of lipid droplets . There iIn the field of cancer metabolism reprogramming and plasticity, we have learned some fascinating lessons. One of the classical examples of metabolic reprogramming is the Warburg effect  which waHowever, developing cancer treatments by targeting altered lipid metabolism is in its infancy. It remains challenging, primarily due to an incomplete understanding of the mechanisms that regulate lipid synthesis, esterification, lipolysis and lipophagy in cancer cells , 113. InWhat is beyond question is the continued lack of life-prolonging treatment for glioblastoma patients despite significant advances in genetic and molecular characterization and almost twenty years since the introduction of temozolomide as the standard of care. This necessitates the discovery of nuanced avenues to understand glioblastoma formation to overcome therapeutic resistance. Ferroptosis and lipid metabolism offer some very early promise in this direction and are worthy of further exploration in this lethal cancer.Although monitoring the balance of PUFAs and MUFAs is known to predict the sensitivity of cells to ferroptosis, cellular sensing and the feedback mechanism that balances PUFAs/MUFAs remain to be investigated.There is imputes to define the relationship between lipid droplets, fatty acid metabolism, and ferroptosis. However, key protein mediators of these biological processes remain to be discovered.The most important factors for ferroptosis-dependent cell death are lipid peroxides that self-propagate along the plasma membrane and result in the accumulation of oxidatively damaged lipids. Our understanding of the downstream pathways of peroxidized phospholipids that execute ferroptosis and potentially contribute to tumorigenesis remains at a nascent phase. The question remains whether there is a repair or antagonism mechanism of peroxide lipids at damage loci in the cell membrane. Are oxidatively damaged membranes prone to recycling? Can this metabolic plasticity contribute to glioblastoma therapeutic resistance? How do these factors cooperate their functions in cancer microenvironments?Many cancers, including glioblastoma, consist of both stem and nonstem cells; however, the degree to which these cells are resistant to ferroptosis remains mostly unknown.Conceptualization, AB-G. Writing-original draft preparation, AB-G, JD, and MW. Review and editing, AB-G, JD, and MW. Funding acquisition, AB-G. All authors have read and agreed to the published version of the manuscript.


Prostate-specific membrane antigen positron emission tomography (PSMA PET) is a modern imaging modality used in the management of patients with prostate cancer with improved accuracy in detecting lymph nodes and distant disease spread. In this paper, we discuss how the increasing use of PSMA PET is changing clinical management in patients with prostate cancer, specifically those previously treated for localized disease and now presenting with recurrence or low-volume metastatic disease spread. We also discuss how PSMA PET is affecting clinical trial design and interpretation. More clinical trials are needed to investigate whether the use of PSMA PET translates into improved patient survival or quality of life.Prostate-specific membrane antigen (PSMA) positron emission tomography (PET) scans have higher sensitivity and specificity for detecting lymph nodes or metastatic disease relative to conventional imaging in prostate cancer staging. Since its FDA approval and incorporation into treatment guidelines, the use of PSMA PET has increased in patients undergoing initial staging, those with recurrence after initial definitive treatment, and patients with metastatic disease. Although the early detection of metastatic lesions is changing disease management, it is unclear whether this impact on management translates into clinical benefit. This review will summarize evidence pertaining to the change in patient management due to PSMA PET use and will discuss the implications of PSMA PET on treatment decisions in prostate cancer, particularly in the settings of biochemical recurrence and metachronous oligometastatic disease. Following FDA approval, PSMA PET use was endorsed by the National Comprehensive Cancer Network (NCCN) and the Society of Nuclear Medicine and Molecular Imaging (SNMMI) guidelines [Prostate cancer (PCa) is the most common cancer and the second leading cause of cancer-related deaths in males in the United States with 248,530 new cases and 34,130 deaths in 2021 [ disease ,7,8. PSM of PSMA . 68Ga-PSidelines ,11.Clinicaltrials.gov was used to select ongoing trials of PSMA PET. Tables were created to collect required information and to summarize the available evidence.The influence of PSMA PET on the management of patients with PCa is undergoing extensive investigation. PSMA PET has higher sensitivity and specificity for detecting lymph node or metastatic disease and has a considerable impact on management decisions relative to conventional imaging . Althoug68Ga-PSMA PET increased with PSA. For the PSA categories 0–0.2, 0.2–1, 1–2, and >2 ng/mL, 42%, 58%, 76%, and 95% of the scans, respectively, were positive [While several factors may affect PSA levels , an incrpositive .18F-DCFPyL PET showed improvements in the specificity and positive predictive value when compared with conventional imaging [18F-DCFPyL PET improved disease localization in this cohort of patients [68Ga-PSMA PET in patients with BCR after RT and/or RP and established high positive predictive value, detection rate, and inter-reader agreement for the localization of recurrent prostate cancer with PSMA PET [Three main studies have established the benefit of PSMA PET over conventional imaging in the recurrent setting. OSPREY is a phase 2/3 trial, which enrolled two cohorts of patients: cohort A included patients with high-risk prostate cancer, and cohort B included patients with metastatic or recurrent disease on conventional imaging.  imaging . CONDOR patients . FendlerPSMA PET . It is, PSMA PET ,46,47,48In patients with BCR after RP, detecting the presence and site of recurrence is crucial for RT planning. Intuitively, the presence of distant disease obviates the need for pelvic or prostate bed RT, and the presence of local failure or lymph nodes has implications for radiation field design, RT dose, and hormone therapy use and duration. Since most SRT is delivered at relatively low PSA levels (<1 ng/mL), conventional imaging is unlikely to add any valuable information for treatment planning, and thus most patients would receive SRT. The ability of PSMA PET to detect disease at low PSA levels prior to SRT offers a unique opportunity for personalized therapy. This has been shown in several studies. In their retrospective analysis, Bottke et al. evaluated the impact of PSMA PET on SRT planning in 76 patients with BCR after RP . PSMA PE18F-DCFPyL PET identified extra-prostatic disease in twice as many men and detected a site of recurrence in 87% of men, compared with 67% with conventional imaging. Furthermore, 18F-DCFPyL PET identified potentially actionable disease (prostate-only recurrence or oligometastatic disease) in 75% of men and changed the proposed management in 43% of men [Compared to surgery, fewer studies have investigated the effect of PSMA PET on the management of patients treated initially with RT as the primary definitive treatment and presenting with BCR. In a prospective study, Liu et al. investigated the influence of PSMA PET compared to conventional imaging in managing patients with radio-recurrent prostate cancer. % of men . Afaq et% of men . Barbaud% of men . Interes% of men ,36,50. T% of men . Several% of men ,38,39.18F-Fluciclovine-PET-based post-prostatectomy RT planning significantly improved survival free from biochemical recurrence or persistence, compared to conventional imaging [18F-Fluciclovine-PET. Randomized trials looking at outcomes in patients whose management is planned with PSMA PET vs. standard of care imaging are ongoing to determine whether these changes are beneficial or not, namely in terms of survival and quality of life.To summarize, PSMA PET influences management in patients with BCR following both RT and RP despite the heterogeneity and retrospective nature of the studies and the variation in the definition of “treatment change” among the different studies. In a meta-analysis of 15 studies, including 1163 patients in the recurrent and definitive settings, the pooled proportion of management changes was 54% with half of these changes entailing a new treatment modality . Althoug imaging . DespiteOligometastatic prostate cancer  includes patients who present with recurrence after initial definitive therapy with imaging showing limited metastatic involvement , and patients presenting with de novo limited metastatic disease . The distinction between synchronous and metachronous metastases is important as they may have different biologic and clinical characteristics. For the purpose of this paper, we will focus on metachronous oligometastatic disease. Whether OMPC is truly a distinct intermediate state between localized and widespread metastatic disease that is less aggressive and less likely to further metastasize is yet to be determined . No consTwo studies have defined the use of radiotherapy, specifically stereotactic body radiotherapy or SBRT, in patients with metachronous OMPC: STOMP and ORIOLE. The randomized phase II ORIOLE clinical trial compared outcomes of men with hormone-sensitive OMPC treated with metastasis-directed therapy (MDT) vs. observation . MDT sigWhile the data are promising, these studies have several limitations. Namely, most of these studies are retrospective in nature, hence prone to a wide range of biases probably leading to an over-estimation of the effect size. Furthermore, patient and treatment characteristics are heterogeneous both among studies and within the same study. Caution is thus warranted in interpreting the results. Given all this information, PSMA PET potentially holds appreciable benefits in guiding MDT for patients with OMPC, and thus delaying the initiation of ADT or delaying the switching of systemic therapy at progression. Again, however, whether this has an impact on outcomes remains poorly understood and should be further investigated.Most of the current recommendations for the treatment of patients with prostate cancer are derived from older studies, before the widespread availability of PSMA PET. The increasing use of PSMA PET has caused a landscape shift and has led to new paradigms and disease presentations, which were not appreciated in the era of conventional imaging. Therefore, while findings are promising, it is still unclear how PSMA PET should change management and how to integrate these advances into modern clinical practice and future trial design. Interestingly, PSMA PET is having a significant impact on the interpretation of endpoints in clinical trials. For example, it is unclear how progression on PSMA PET should be exactly defined and whether it is clinically meaningful, especially when considering that PET-based metastatic progression happens before progression on conventional imaging. In patients enrolled on clinical trials, PSMA PET-based interventions may prolong conventional imaging-based metastasis endpoints and make interpretations of trial results difficult. Along the same lines, it is unclear whether progression on PSMA PET should lead to a change in therapy, such as adding or switching systemic therapies or adding MDT. Although a proposal for systemic therapy response–assessment criteria with PSMA PET has been published , more womax are low, when there is only one lesion on the scan , or when there is an absence of CT correlates. Such findings should raise suspicion for a false positive.While not discussed in this review, PSMA PET imaging is indicated per the NCCN guidelines in the initial staging of patients with unfavorable intermediate- and high-risk disease. PSMA PET could be used for dose escalation to avid lesions, as per the FLAME study (which used MRI rather than PSMA PET) ,78, giveThe increased sensitivity of PSMA PET leads to stage migration as patients previously thought to be at a low stage are upstaged based on new PSMA PET findings. This leads to improved survival in both stages: survival in the lower stage improves as the more advanced patients are upstaged and survival in the higher stage also increases when favorable patients enter this group. It is unclear how to exactly deal with stage migration when deciding on treatments, especially in patients considered for clinical trials. For example, how do we manage patients with BCR after prior RP and negative conventional imaging but with one small distant lesion on PSMA PET? Is biopsy always indicated in these situations? Should these patients receive salvage RT to the pelvis, or should they be treated with systemic therapy? If so, what is the most appropriate systemic therapy? Additionally, should they receive MDT? Finally, could such patients be enrolled on a trial of salvage radiotherapy that does not mandate molecular imaging? Currently, the clinical trial NRG GU-008, which randomizes patients with nodal involvement after prostatectomy to salvage radiation and long-term ADT with or without Apalutamide, excludes patients with M1 disease on molecular imaging. Similarly, it is unclear how to manage definitive patients with PSMA PET findings which are not corroborated by conventional imaging. Interestingly, the currently enrolling NRG GU-009 and NRG GU-010 allow patients who have bone metastases established only by Fluciclovine, Choline, or PSMA PET but are not definitive on bone scan or NaF PET to enroll. Finally, it is not clear how to specifically treat patients with metachronous OMPC with bone lesions per PSMA PET. Options include ADT alone, ADT with additional androgen receptor (AR)-targeted agents, MDT alone, MDT and ADT, or MDT and ADT plus additional AR-targeted agents. Currently, there is extreme heterogeneity in how these patients are being treated clinically, including patients progressing on clinical trials, which will make the future interpretation of data difficult. NRG GU-011 is trying to answer one part of this question.This review has several limitations. First, we did not follow a systematic approach for the selection of papers as in a systematic review or meta-analysis. This means that there is the potential for selection bias. Second, we did not formally evaluate bias or heterogeneity. Third, the cohorts of patients included are not homogeneous. Although each section deals with a general presentation of disease , each study had its own inclusion and exclusion criteria, as well as its own definition of recurrence or OMPC. Thus, caution is warranted in drawing conclusions. Fourth, most of the included studies are retrospective in nature, and thus are inherently prone to certain biases, such as overestimation of effect size. Although a more systematic review should be conducted in the future, our work nonetheless provides readers with a summary  of a large body of evidence relating to the effect of PSMA PET on management decisions. Finally, while we group all PSMA PET imaging modalities together, it is important to discuss the advantages and limitations offered by PSMA PET CT versus PSMA PET MRI. PSMA PET MRI combines the molecular data from PET with the superior anatomic details of the MRI. Although PSMA PET MRI shows good diagnostic performance, there is no clear data showing its superiority over PET CT . PET CT 177Lu-PSMA-617) show clinical benefit, the underlying resistance mechanisms and the role of PSMA PET imaging as a biomarker for patient selection will need to be further investigated in future studies [As previously discussed, most of the available literature is retrospective in nature, severely limiting reliable conclusions. Several prospective trials are currently underway evaluating the role of PSMA PET imaging in the management of prostate cancer at initial staging, recurrence, and in (oligo)-metastatic disease. Importantly, many of these studies are comparing oncologic outcomes between standard of care vs. molecular imaging. A selected list of ongoing trials is presented in  studies ,87,88,89In conclusion, PSMA PET provides many answers and important information for clinicians when it comes to the management of patients with prostate cancer even at low PSA values where traditional imaging modalities are completely inadequate at detecting malignant lesions. However, PSMA also raises many questions, which remain unanswered. Larger randomized trials are needed to truly validate the effect of PSMA PET on patient outcomes in different disease settings.


Cancer recurrence and drug resistance following treatment, as well as metastatic forms of cancer, are trends that are commonly encountered in cancer management. Amidst the growing popularity of personalized medicine and targeted therapy as effective cancer treatment, studies involving the use of stem cells in cancer therapy are gaining ground as promising translational treatment options that are actively pursued by researchers due to their unique tumor-homing activities and anti-cancer properties. Therefore, this review will highlight cancer interactions with commonly studied stem cell types, namely, mesenchymal stroma/stem cells (MSC), induced pluripotent stem cells (iPSC), iPSC-derived MSC (iMSC), and cancer stem cells (CSC). A particular focus will be on the effects of paracrine signaling activities and exosomal miRNA interaction released by MSC and iMSCs within the tumor microenvironment (TME) along with their therapeutic potential as anti-cancer delivery agents. Similarly, the role of exosomal miRNA released by CSCs will be further discussed in the context of its role in cancer recurrence and metastatic spread, which leads to a better understanding of how such exosomal miRNA could be used as potential forms of non-cell-based cancer therapy. Over the past decade, registered clinical trials involving the use of MSC, IPSC, and CSC in cancer therapy have grown, and more recently, iMSC was added to the list. In the midst of the controversial reputation of MSC cells in the world of cancer studies, the therapeutic roles such as their tumor-homing capabilities, paracrine signaling activities, and delivery of exosomal microRNA (miRNA) cargo are well-known mechanisms employed by these cell types in communicating with cancer cells within the tumor microenvironment (TMC). In addition, with more reliable iPSC generated through non-viral and non-integrated reprogramming platforms, a breath of new options for more stable pluripotent cells and their derivatives are obtainable. One such candidate is MSC generated though differentiation of fully reprogrammed cells (iMSC) which may potentially offer another alternative cell type that compensates for the heterogeneity and scalability issues of MSCs while maintaining all the valuable therapeutic properties of MSCs. IPSCs have also been shown as feasible disease models for understanding cancer progression and their resistance to cancer drugs. Finally, by understanding the plasticity of CSC in tumor progression and metastasis, and how exosomal miRNA plays a pivotal role in the cancer stem cell niche, potential anti-cancer candidates could be developed, leading to non-cell-based cancer therapy. Therefore, this review focuses on the current perspective of how major stem cell types interact with cancer cells and their recent progress toward the development of cell-based and non-cell-based cancer therapy.Human MSCs are heterogenous populations of multipotent stem cells that can be obtained from different origins, and among the most commonly studied are the bone marrow, adipose tissue, umbilical cord, Wharton’s jelly, dental pulp, and peripheral blood mononuclear cells . The intWhile studies have shown the various roles of MSCs in cellular functions, including the ability to differentiate into various cell types for damaged tissue repair and regeneration, current evidence has also pointed to the paracrine signaling mechanism ,5 relateThe dual role of MSC in cancer-related studies, as either promoting or suppressing cancer activities in cancer cells, is a much-accepted fact, with various reports linking MSC as a tumor enhancer via mediating angiogenesis, attenuating immune reactions, initiating epithelial to mesenchymal transition (EMT), and promoting metastatic processes whereas the tumoricidal effect of MSC on cancer cells appears to be more apparent through the induction of apoptosis and signaling pathways alterations .Several studies have revealed that MSCs can support the tumor vasculature via direct differentiation of MSCs into myofibroblasts that are were then transformed into tumor-associated fibroblasts (TAFs)  or indirMesbah et al. (2021) showed that the cultivation of colorectal cancer cells with MSC inhibited proliferation and induced apoptosis of colorectal cancer cells with increased expressions on P53, Caspase3, and P21. The tumoricidal or pro-neoplastic effect of MSC on cancer can also be attributed to its hypo- or hyper-activation of certain signaling pathways associated with cancer proliferation, survival, and progression . Among tAnother exciting development at the forefront of MSC and cancer therapy is the interaction between exosomal miRNA and cancer cells. The dysregulation of exosomal miRNA was shown to promote tumor growth due to the activation of angiogenic signaling pathways such as VEGF , hedgehoDue to the homing properties of MSCs towards the tumor site, recent studies have focused on utilizing MSCs as carriers for anti-cancer agents, including MSC expressing IL-18, TRAIL, oncolytic adenovirus (CRAd5/F11), and paclitaxel-encapsulated nanoparticles in the treatment of breast cancer cells , B-cell Although a big proportion of trials registered on ClinicalTrials.Gov were designed to evaluate MSCs that are not genetically modified, limited data have since been published from such trials. One such published study described the role of allogenic bone marrow-derived MSC in localized prostate cancer patients to assess the safety and feasibility of MSC-homing capacity to localized prostate cancer regions (NCT01983709) and revealed that MSCs were as well tolerated with no dose-limiting toxicities observed. However, as MSCs were not detected to home primary tumors in any of the study subjects, the trial was terminated . NeverthFurthermore, exosomes have been shown to be abundantly produced in MSCs , and exoThe dual roles of MSC to either suppress or promote tumor development could be attributed to the variations in the tumor model utilized among different studies, tissue source of MSC, epigenetic variability as well as the heterogeneity of the isolated MSCs., In addition, the timing and dosage of MSC administration, variations in cellular delivery methods and, culture conditions utilizing different supplements were commonly shown to influence the interaction between MSCs and cancer cells ,63. FurtiPSCs were first discovered in 2007 by , wherebyCommon issues that affect the clinical translation of using MSC in cancer therapy are its heterogeneity and difficulty of scalability . Hence, Several in vitro and in vivo studies have been carried out to test the impact of iMSC on multiple cancers including breast  and lungFurther studies with genetically engineered iMSC revealed promising therapeutic effects on different cancers ,83,84. LNotably, extracellular vesicle (EV) mimics made from iPS cell-derived mesenchymal stem cells were shown to improve the treatment of metastatic prostate cancer  and geneiPSCs can be generated from malignant cancer cells as a live human cell model to evaluate the stages of oncogenesis, which starts with cellular transformation followed by the hierarchical organization of established malignancies. Also, reprogramming cancer cells could aid in determining the interaction among the genetic and epigenetic drivers of carcinogenesis. In a colorectal cancer model, iPSCs-derived colorectal cancer organoids were developed to test drug sensitivity, revealed that geneticin was able to suppress WNT signaling activation, rescue adenomatous polyposis coli (APC) protein expression levels, and suppressed the colonic epithelial proliferation in familial adenomatous polyposis colonic organoid model . In anotAlternatively, iPSCs may serve as another avenue for adoptive immune therapy via its deviation into natural killer cells (iPSCs-NK). These iPSCs-NK cells could be engineered via knock-in/out of genes resulting in the generation of allogenic iPSCs-NK cells capable of suppressing MHC1 leading to avoidance of host T cell recognition. These engineered cells also over-expressed HLA-E, resulting in the inactivation of host NK cells . The repCancer stem cell (CSC) is a group of highly dysregulated cancer cells with abnormal karyotyping, unlimited capacity to self-proliferate, highly aggressive, and possesses abilities to resist different types of anti-cancer therapies ,95,96,97The TME in breast cancer consists of cancer cells, immune cells, stromal cells, and various local factors originating from either the local breast cancer or distant tissues . In breaIn colorectal cancer, tumor-infiltrating cells, extracellular matrix (ECM), blood vessels, and other matrix-associated substances make up the TME . As a keHigh-grade glioma is a highly aggressive brain tumor  and its The TME in liver cancer typically consists of cancerous cells, stromal cells, vasculature and immune cells that form a microenvironment that promotes cancer cell proliferation, migration, and treatment resistance . In liveLike other solid tumors, TME does exist in hematological malignancies and TME in hematological malignancies typically contain various hematological cells, immune cells, and local factors that form a proliferative niche with enhanced pro-tumoral activities . SeveralIn summary, circulating CSC-regulating miRNAs could serve as double-edged swords in either promoting or suppressing cancer development ,111,112.https://clinicaltrials.gov/) (accessed on 30 November 2022). Among these studies, two trials (NCT01577511 and NCT02052908) have focused on colorectal cancer, while one trial has focused on AML (NCT01298414), breast cancer (NCT01231386), glioblastoma (NCT05328089), and melanoma (NCT01216787), respectively. Four out of the six studies have been completed, while one study (NCT05328089) is still recruiting participants, whereas one trial has been terminated because of unclear reasons. Some of these studies are aimed at identifying specific circulating miRNA expression profiles in the cancer stem cells , and some other studies  are working to delineate specific profile changes in the circulating miRNAs after an interventional therapy was administered.To date, at least six clinical trials  have aimCirculating exosomal or EV-encapsulated miRNAs have great potential in mediating the CSC phenotypes in different types of cancers ,97,132. There is no doubt that the knowledge gained from studies involving MSCs, iMSC, iPSCs, and CSC with the tumor microenvironment has enhanced our understanding of their unique stem cell properties, often showing plasticity in response to their surrounding environment. Despite the risk of propagating the side effects of cancer progression or tumor growth, these stem cells have also shown to be a promising anti-cancer agent especially through the natural paracrine signaling and exosomal miRNA interaction with cancer cells. However, there still exist many uncertainties on how these stem cells and their secretome cause a direct influence on inhibiting tumor growth or cancer progression to a metastatic state. Nevertheless, numerous findings of in vitro cell studies and clinical trials have provided a way forward with the prospect of using targeted approaches such as exosomal miRNA as an alternative to cell-based cancer therapy to generate more specific positive outcomes as well as bioengineering methods for a more safer and specific delivery of cell- and non-cell-based therapy along with overcoming scalability and heterogeneity of stem cells. These are certainly elements of promising avenues that garner a strong case for incorporating and leveraging on the available knowledge and technological advancement in the area of stem cell research and their feasible application in paving a sustainable approach towards generating good manufacturing practices (GMP) grade cancer therapies capable of delivering the appropriate anti-cancer drug in a more targeted manner with precision effect on cancer treatment.


The multidisciplinary meeting (MDM) has been endorsed in current international consensus guidelines as the gold standard method for diagnosis of interstitial lung disease (ILD). In the absence of an accurate and reliable diagnostic test, the agreement between multidisciplinary meetings has been used as a surrogate marker for diagnostic accuracy. Although the ILD MDM has been shown to improve inter-clinician agreement on ILD diagnosis, result in a change in diagnosis in a significant proportion of patients and reduce unclassifiable diagnoses, the ideal form for an ILD MDM remains unclear, with constitution and processes of ILD MDMs varying greatly around the world. It is likely that this variation of practice contributes to the lack of agreement seen between MDMs, as well as suboptimal diagnostic accuracy. A recent Delphi study has confirmed the essential components required for the operation of an ILD MDM. The ILD MDM is a changing entity, as it incorporates new diagnostic tests and genetic markers, while also adapting in its form in response to the obstacles of the COVID-19 pandemic. The aim of this review was to evaluate the current evidence regarding ILD MDM and their role in the diagnosis of ILD, the practice of ILD MDM around the world, approaches to ILD MDM standardization and future directions to improve diagnostic accuracy in ILD. Interstitial lung disease (ILD) refers to a diverse group of disorders characterized by varying degrees of inflammation and/or fibrosis of the lung parenchyma . Due to Multidisciplinary discussion has been considered the gold standard method for diagnosis of ILDs for the past two decades. The ILD multidisciplinary meeting (MDM) involves dynamic discussion between different subspecialists whereby all available case details are carefully reviewed, and a consensus diagnosis reached. Previously, the findings obtained from surgical lung biopsy were considered the gold standard for ILD diagnosis. The MDM was first recommended to replace histopathology as the gold standard in the 2002 American Thoracic Society (ATS)/European Respiratory Society (ERS) Joint Statement on the Classification of Idiopathic Interstitial Pneumonias . This reDespite strong support for the MDM from expert bodies, there is a paucity of evidence regarding ideal ILD MDM composition. Additionally, reduced access to specialist ILD centers impacts patients and clinicians in many parts of the world. Despite these challenges, it has been demonstrated that a multidisciplinary approach to ILD diagnosis has been widely adopted in routine care settings globally . The absInterstitial lung diseases place enormous encumbrance on patients, carers, and health care systems. Many patients with ILD are at risk of progressive fibrotic disease, associated with reduced quality of life and premature mortality. Idiopathic pulmonary fibrosis (IPF), the most common fibrotic ILD is almost universally progressive and up to 40% of non-IPF ILD are also observed to progress . Both eaFor these reasons, early and accurate diagnosis is critical. ILD MDMs, involving case discussion between health professionals from different specialties to generate a consensus diagnosis for the patient, aim to maximize available clinical data, expertise and therefore, diagnostic accuracy. Traditionally, the ILD MDM is chaired by a respiratory physician with expertise in ILD. Other important contributors include other respiratory physicians, radiologists and histopathologists with expertise in ILD, rheumatologists and immunologists, ILD nurses, trainees, and other health professionals. Availability of resources will dictate the constitution of an MDM at each site. In addition to generation of a consensus ILD diagnosis, the ILD MDM also functions as a forum to consider patient prognosis and management.Since its implementation, a multidisciplinary approach has consistently been associated with higher levels of diagnostic confidence, better inter-observer agreement and lower rates of unclassifiable diagnoses—considered surrogate markers for diagnostic accuracy , 20. Forw] = 0.65, IQR 0.53–0.72) than non-expert physicians  and physicians without access to an ILD MDM demonstrated the lowest rate of inter-observer agreement . Importantly, association with an academic hospital or university, longer duration of ILD experience and access to an ILD MDM were all independently associated with greater prognostic accuracy of IPF diagnosis, thus demonstrating the clinical benefit of having experienced physicians present at the ILD MDM and training of non-experienced clinicians ] and connective tissue disease-associated ILD (CTD-ILD) [κw = 0.72 (0.68–0.78)]; however, only moderate for non-specific interstitial pneumonia (NSIP) [κw = 0.42 (0.37–0.49)], and fair for hypersensitivity pneumonitis (HP) [κw = 0.29 (0.24–0.40)].The largest multicenter evaluation of diagnostic concordance between different ILD MDMs to date was conducted by Walsh et al. in 2015 . DiagnosVarious reasons have been proposed as to why such discordance exists , 28–33. Importantly, factors relating to MDM structure, organization and administration, governance and clinical decision-making processes are likely to differ between MDMs. Jo et al.  surveyedvia electronic questionnaires showed 79.6% held formal ILD MDMs to discuss patient diagnosis and management , hold solely face-to-face meetings (83.6 vs. 73.3%), have meetings of between 31 and 60 min duration (52.4 vs. 33.3%) and have at least four disciplines in attendance (60.8 vs. 33.3%) compared with participating centers in lower-middle income countries . AlthougAlthough up to 20% of ILDs are associated with underlying CTDs such as systemic sclerosis, rheumatoid arthritis and idiopathic inflammatory myopathies, rheumatologists and immunologists are not routinely involved in ILD MDMs , 38. In A recent observational study from a large Italian ILD expert center analyzed consecutive ILD MDM cases with suspected new diagnosis or progression of CTD-ILD, concluding that involvement of a rheumatologist in the MDD resulted in availability of more comprehensive clinical information and improved diagnostic accuracy .Another recent Italian study involving a Delphi survey and additional questionnaires distributed to pulmonologists, rheumatologists and radiologists demonstrated high levels of agreement regarding the importance of a collaborative approach to diagnosis of CTD-ILD . ResultsDespite scarcity of supporting evidence, expert panels have suggested approaches to ILD MDM organization in attempts to provide a standard framework for universal implementation . As a miAn example of a standardized framework for the ILD MDM is the 2017 Thoracic Society of Australia and New Zealand and the Lung Foundation Australia Position Statement on the ILD MDM and accompanying ILD toolkit, consisting of practical material designed to aid in the presentation and discussion of cases at ILD MDMs , 411 ThiIt is also worth noting the proliferation of interstitial lung disease registries worldwide in the last 20 years, such as the Australasian ILD Registry (AILDR) , the PulWorldwide standardization of the ILD MDM will require an understanding of factors contributing to diagnostic heterogeneity as well as consensus regarding the purpose and desired outcomes of multidisciplinary discussion. Recently, Teoh et al.  conducteExperts strongly agreed upon five essential features for an ILD MDM, including: 1) the need for at least one radiologist, 2) high-quality HRCT images for each case, 3) technological infrastructure enabling real-time viewing of CT scans, 4) a quiet environment enabling uninterrupted, free-flowing discussion and 5) a standardized template for documentation of outputs from each case discussion. Additionally, experts agreed upon several other desirable components as outlined in Many questions remain. Notably, there was no consensus agreement upon the need for attendees from other specialty types such as rheumatology or immunology; and although a histopathologist was felt to be “highly desirable”, their presence was not considered essential. This likely reflects limited access, particularly in smaller and more remote centers. The authors also noted that viability is a concern, with ongoing increases in the workload of ILD MDM clinicians, radiologists, and pathologists, limiting time and resources available to dedicate to consideration of each available case. Of note, the pathologist is a key contributor in only a small fraction of cases where a biopsy has been performed. Additionally, in this modern era, lack of onsite pathology does not preclude involvement in the MDM as tissue pathologists can participate virtually even if a center does not have an onsite pathologist.Interestingly, ILD experts agreed that research terminologies such as IPAF could also be documented as present by consensus at MDM. Although this term has not been validated as a distinct diagnostic entity, its use may be appropriate where the ILD MDM's favored diagnosis is suspected CTD-ILD, rather than idiopathic interstitial pneumonia, but the patient doesn't satisfy CTD diagnostic criteria. However, further studies using refined IPAF criteria are likely required before IPAF can be considered a distinct diagnosis and to inform evidence-based management of affected patients.via videoconferencing or web-based platforms, or a hybrid model, is best. Nonetheless, these consensus agreements on the ideal features of an ILD MDM are fundamental in informing future research into MDM standardization.The ideal format of an ILD MDM also remains uncertain—whether meeting entirely face-to-face, virtually Recent scientific and technological developments have had significant impact on both the inputs and outputs of the ILD MDM. Expanded therapeutic options and an increasing array of clinical trials have added to the complexities of management. Furthermore, the global pandemic has necessarily transformed the essence of human interaction, particularly in the healthcare setting. Some key recent influences on the ILD MDM are considered below.Evolution of clinical practice guidelines for diagnosis of ILDs and the availability of the antifibrotic drugs nintedanib and pirfenidone have been associated with increased frequency and complexity of referrals to ILD specialist centers for MDM consideration . ChangesIn these settings, it can be tempting to label patients as having IPF or PPF despite an alternative leading differential diagnosis in order to overcome the limitations of regulatory prescribing. Although this is a pragmatic strategy, it is worth acknowledging the risks associated with such practice. With respect to the “PPF” (or progressive fibrosing ILD “PF-ILD”) label in centers where antifibrotics are available for this indication, “lumping” a heterogeneous group of patients together, rather than “splitting” into specific diagnostic groups risks limiting opportunities to identify inflammation-driven disease activity or other causes of deterioration. International guidelines emphasize the treatment for PPF must be agnostic to the underlying condition and all efforts must be made to procure and treat a leading diagnosis before labeling it as progressive .TBLC has recently been demonstrated in both clinical trials and meta-analyses to be a reliable method for lung tissue sampling for histopathologic assessment and MDM diagnosis in ILD; a less invasive procedure with lower rates of complications such as pneumothorax and airway bleeding compared with traditional surgical lung biopsy although the quality of evidence is low –52. The The recently updated ATS/ERS/JRS/ALAT clinical practice guideline for IPF and PPF  includesThere has been significant recent research interest into novel tools to increase diagnostic yield and accuracy in ILD diagnosis, including the use of genomic classifier testing .Importantly, there is a growing understanding of pathogenic mutations linked to an inherited risk of pulmonary fibrosis. Mutations in telomere-related genes such as telomerase reverse transcriptase (TERT) have been identified in up to one-quarter of familial pulmonary fibrosis patients and have been associated with the “short telomere syndrome”, predisposing affected individuals to progressive ILD, premature hair graying, cryptogenic liver cirrhosis, hematological abnormalities, and reduced survival , 56. DesIntegration of genetic data into the ILD MDM is yet to be validated, yet research is underway. A recent international survey including 352 respiratory physicians demonstrated support for the use of genetic testing in ILD, predominantly in view of its impact on diagnostic investigation approach and patient treatment, however 88% identified a need for more information on its role and interpretation of results .Currently, genomic testing might be considered within the MDM for patients with a family history or atypical disease presentation, to help predict expected disease behavior and guide management discussions. For example, patients with inherited or sporadic ILD-associated genetic mutations might be recommended for earlier consideration of lung transplant referral, and avoidance or caution with immunosuppressive therapy. It could also be recommended with a view to potential clinical trial participation, such as current phase II studies into the use of the androgen danazol for treatment of familial pulmonary fibrosis associated with short telomere length . AdditioTM genomic classifier, which employs a machine learning algorithm developed to classify usual interstitial pneumonia (UIP) vs. non-UIP ILD patterns, uses bulk RNA-sequencing data obtained from high throughput sequencing of exome-enriched RNA extracted from transbronchial lung biopsies or TBLC .LG has received travel and conference support from Boehringer Ingelheim. LT has provided paid consultancy for Erbe Elektromedezin and Boehringer Ingelheim. TC has received grant support, consultancy fees, and speaking honoraria from Boehringer Ingelheim and Hoffman-La Roche, consultancy fees from Bristol Myers Squibb; grant support from Biogen, and provides consultancy for DevPro and Ad Alta.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Microscopic and spectroscopic techniques are commonly applied to study microbial cells but are typically used on separate samples, resulting in population-level datasets that are integrated across different cells with little spatial resolution. To address this shortcoming, we developed a workflow that correlates several microscopic and spectroscopic techniques to generate an in-depth analysis of individual cells. By combining stable isotope probing (SIP), fluorescence in situ hybridization (FISH), scanning electron microscopy (SEM), confocal Raman microspectroscopy (Raman), and nano-scale secondary ion mass spectrometry (NanoSIMS), we illustrate how individual cells can be thoroughly interrogated to obtain information about their taxonomic identity, structure, physiology, and metabolic activity. Analysis of an artificial microbial community demonstrated that our correlative approach was able to resolve the activity of single cells using heavy water SIP in conjunction with Raman and/or NanoSIMS and establish their taxonomy and morphology using FISH and SEM. This workflow was then applied to a sample of yet uncultured multicellular magnetotactic bacteria (MMB). In addition to establishing their identity and activity, backscatter electron microscopy (BSE), NanoSIMS, and energy-dispersive X-ray spectroscopy (EDS) were employed to characterize the magnetosomes within the cells. By integrating these techniques, we demonstrate a cohesive approach to thoroughly study environmental microbes on a single-cell level. Efforts to understand the ecology of mixed environmental microbial populations are often hindered by our inability to collectively study the morphology, physiology, and taxonomy of distinct taxa on a meaningful scale. By correlating different single cell resolving spectroscopic and microscopic methods onto a single sample, more meaningful ecophysiological interpretations can be made. Cultivation-independent techniques such as microautoradiography and nano-scale secondary ion mass spectrometry (NanoSIMS), as well as next-generation physiology approaches such as Raman microspectroscopy (Raman), have been used to study the in situ function and interactions of uncultured microbes . In conjCorrelative microscopy was developed as a means to overcome the limitations in resolution of conventional light microscopy by combining fluorescence microscopy (FM) with electron microscopy (EM), thus allowing for ultrastructural features within eukaryotic cells to be studied in greater detail , 5. The Here, we illustrate a workflow to correlate FISH, SEM, Raman, and, if desired, NanoSIMS to provide a comprehensive characterization of microorganisms at single-cell resolution. This approach enables the study of the taxonomic identity (rRNA-targeted FISH), morphology (SEM), biochemistry (Raman), and metabolic activity (SIP-Raman and/or SIP-NanoSIMS) of cells within microbial communities or phenotypic heterogeneity within a clonal population of cells. This is, to our knowledge, also the first correlative application of both Raman microspectroscopy and NanoSIMS on the same cells.Escherichia coli and the archaeon Methanosarcina acetivorans. Next, we applied the correlative workflow to multicellular magnetotactic bacteria , which are affiliated with the bacterial phylum Desulfobacterota (previously described as the class Deltaproteobacteria . As. As3S4; tra Fig. .Fig. 3Co2O could not be detected via Raman, NanoSIMS showed that all MMB analyzed (n = 23) exhibited low levels of deuterium incorporation . Group 4 was the second most abundant , and Group 3 was the third most abundant . Unlabeled MMB accounted for 23% (n = 22) of all MMB and likely belonged to Groups 2 and 5 that were not targeted in this study. Our findings on the relative abundance of different MMB populations are consistent with the results reported by Simmons et al., who studied an unspecified tidal pool in LSSM [The three MMB populations exhibited similar morphology when studied via FE-SEM, indicating that different MMB species found in LSSM cannot be distinguished by morphology alone Fig. , highlig in LSSM .3S4) in the Raman spectra of MMB  Both BSE-SEM and EDS were applied outside of the correlative workflow due to the negative effect of high voltage electron beams on cells. MMB that had been analyzed using BSE-SEM and EDS could not be labeled using FISH, and the respective Raman spectra were consistent with carbonized biomass, likely due to the effect of using a high voltage electron beam (not shown). Previous correlative studies have used low-dose electron imaging, such as FE-SEM, to avoid irreversible damage to the cell caused by high electron voltages .Understanding the metabolic potential and ecological distribution of microbial communities has been greatly aided by various omic techniques, but information on the morphology, physiology, and taxonomy of distinct taxa is lost during these analyses. Here, we provide a single cell level workflow to link the taxonomic identity and morphology of microbes using rRNA-targeted FISH and SEM with their cellular chemistry and metabolic activity using SIP-Raman and/or SIP-NanoSIMS. By applying this workflow, naturally low abundance populations  can be taxonomically identified and interrogated in greater detail than current practices allow. Furthermore, having correlated data is desirable for population studies to determine if physiological heterogeneity exists within clonal groups of cells.2O as a general marker of anabolic activity, but substrates labeled with 13C, 15N, 18O, or 34S could be incorporated in the workflow. Additionally, future applications and developments of this workflow might include the use of substrate analog probing [In this study, we used D probing , 65, or  probing , nucleic probing , or lipi probing , its use probing  and prot probing , 69 coulSupplemental InformationSupplemental Table


Early appropriate empirical antibiotics are critical for reducing mortality in sepsis. For hospital-acquired sepsis of unknown origin in Australia, piperacillin-tazobactam (TZP) is recommended as an empirical therapy. Anecdotally, some institutions also use TZP for community-acquired septic shock. This narrative review aimed to scrutinise the appropriateness of TZP as an empirical agent for undifferentiated hospital-acquired sepsis and community-acquired septic shock. An online database (Medline) was searched for relevant studies in adults published in the last 10 years. Studies were included if they addressed separately reported clinical outcomes related to a relevant aspect of TZP therapy in sepsis. Of 290 search results, no studies directly addressed the study aim. This review therefore explores several themes that emerged from the contemporary literature, all of which must be considered to fully interrogate the appropriateness of TZP use in this context. This review reveals the paucity and low quality of evidence available for TZP use in sepsis of unclear origin, while demonstrating the urgent need and equipoise for an Australian audit of TZP use in patients with sepsis of unknown origin. Escherichia coli [Staphylococcus aureus, accounted for the remaining 31% of sepsis cases [Sepsis is a serious infectious syndrome caused by myriad pathogens and is a significant contributor to mortality, morbidity, and healthcare costs in Australia and worldwide. Sepsis is defined by The Third International Consensus Definitions for Sepsis and Septic Shock as ‘life-threatening organ dysfunction caused by a dysregulated host response to infection’ . In clinhia coli . Gram-pois cases .Early and adequate empirical antibiotics are a key predictor of outcome in sepsis . The choS. aureus in septic shock or line-related sepsis and substituting TZP for IV meropenem when there are risk factors for multi-drug-resistant (MDR) gram-negative bacteria [TZP is a broad-spectrum IV antibiotic composed of piperacillin, a ureidopenicillin with extended spectrum activity, and tazobactam, a beta-lactamase inhibitor . Piperacbacteria .E. coli and 10.0% of Klebsiella pneumoniae bloodstream isolates had extended-spectrum beta-lactamase (ESBL) phenotype and 8.3% of bloodstream isolates were ‘ESCAPPM’ AmpC producers  [Pseudomonas aeruginosa isolates tested in Australia were resistant to antibiotics of at least two classes [Emerging resistance mechanisms in gram-negative bacteria raise concerns regarding the continued appropriateness of TZP as a first-line broad spectrum antibiotic. In Australia in 2020, 14.7% of species) . In 2020 classes . ESBLs d classes . AmpC be classes . AmpC be classes . The thr classes . Even if classes . TZP is  classes .Important considerations in evaluating TZP appropriateness are toxicity and dosing. Nephrotoxicity and neurotoxicity are potential adverse events reported in the literature, but studies in patients with defined sepsis are limited ,14. ApprThe aim of this review is to address the appropriateness of TZP as an empirical agent for hospital-acquired sepsis and community-acquired septic shock of unknown origin in Australia. Because of a gap in the literature for efficacy of empirical TZP in sepsis of unknown origin in Australia, this review will focus more broadly on clinical outcomes of empirical or definitive TZP for sepsis of any source in any country. Moreover, this review will not separate clinical outcomes related to hospital versus community-acquired sepsis and sepsis versus septic shock, as these terms are often variably and poorly defined in the literature and are rarely analysed as separate entities. Therefore, this review summarises the contemporary literature surrounding clinical outcomes related to empirical or definitive TZP efficacy, toxicity, and dosing considerations in sepsis of any source. Subsequently, these findings are expected to establish the basis of a study addressing the original research question, relating to hospital-acquired sepsis and community-acquired septic shock of unknown origin in the Australian setting.The online database Medline (Ovid) was searched on 13 August 2021 using MeSH terms and key words for ‘sepsis’ and ‘TZP’ and publication date from 2011 to 2021. Search history is listed in full in Written in a language other than English;Meta-analysis or study protocol;Full text unavailable online;The study exclusively concerned a paediatric population;Outcomes relating to either TZP therapy or sepsis were not reported separately from other outcomes;Study participants did not have known or suspected sepsis, as defined by positive blood culture, clinical diagnosis of sepsis, bacteraemia, or bloodstream infection;The study did not address clinical outcomes related to a relevant aspect of TZP therapy; efficacy, resistance, toxicity, or dosing regimens.Studies were excluded on title and abstract and full text screening for the following reasons:Studies were included in the review if a major aim of the study was to investigate clinical outcomes of TZP in sepsis in adults. In order to specifically address the rapidly emerging contemporary evidence on the clinical efficacy of TZP in ESBL and AmpC beta lactamase-producing gram-negative pathogens, an additional search was performed using the key words ‘piperacillin-tazobactam’ and ‘ESBL’ or ‘AmpC’ on 26 April 2022.safetyandquality.gov.au (accessed on 14 August 2021) and included to assess the relevance of international studies in the Australian context. Additional studies referenced only in the introduction were retrieved from the reference lists of included articles.The latest Antimicrobial Use and Resistance in Australia (AURA) and sepsis outcome program reports were retrieved from No studies identified from the database search directly addressed sepsis of unknown origin or the appropriateness of TZP monotherapy in sepsis of any source. One study addressed the appropriateness of TZP/vancomycin combination therapy in sepsis  but was This review addresses the appropriateness of TZP as an empirical agent for sepsis of unknown origin from three angles; clinical efficacy against known pathogens, reported toxicities in septic patients, and optimised dosing.E. coli and K. pneumoniae blood isolates harboured ESBLs [In 2019, more than 1 in 10 Australian ed ESBLs , but evied ESBLs . HoweverEnterobacteriaceae.Harris et al. conducted a noninferiority RCT in nine countries, including Australia, that concluded that definitive TZP, compared with meropenem, was associated with increased 30-day mortality . HoweverOut of eight retrospective observational studies directly comparing TZP and carbapenems in ESBL-PE sepsis, only two found an association between empiric TZP use and mortality ,28. One Proteus mirabilis [In two retrospective studies, lower TZP MIC was associated with reduced mortality in TZP-treated ESBL-PE sepsis than higher MIC, even within the Clinical and Laboratory Standards Institute (CLSI) susceptible range of ≤16 mg/L ,37. Mostirabilis . HoweverApart from the general limitations of retrospective studies discussed later, most studies discussed so far have only included TZP-susceptible infections. Therefore, the available evidence should be applied cautiously to routine clinical practice, where empirical therapy is implemented without knowing the susceptibility profile of the causative organism.Enterobacter and Serratia species but in only 19% of Citrobacter species [A pilot RCT comparing meropenem and TZP for definitive treatment of bloodstream infections caused by ESCAPPM species known to possess inducible chromosomal AmpC beta-lactamases demonstrated no difference in clinical outcomes . Five Am species . The rem species ,18,19. HP. aeruginosa, although this is based on low-quality evidence. Only 5.5% of P. aeruginosa blood isolates were TZP-resistant in Australia in 2020 [P. aeruginosa sepsis susceptible to TZP by disc testing, there was no significant difference in 30-day mortality or clinical/microbiological response between definitive TZP and other anti-pseudomonal agents, including carbapenems and ceftazidime [P. aeruginosa sepsis associated with liver abscesses, after initial resolution of fever with empirical TZP, the patient developed a new liver abscess harbouring TZP-resistant P. aeruginosa [TZP appears to be a generally effective agent for sepsis caused by TZP-susceptible  in 2020 . Reassur in 2020 . Moreovetazidime . As withruginosa .Eggerthella lenta is suspected. A retrospective cohort study of E. lenta sepsis found high MICs to TZP and an independent association with 30-day mortality and Intensive Care Unit (ICU) stay in 43 patients treated with empirical TZP [Several case reports suggest that TZP is an appropriate antibiotic for less common pathogens involved in anaerobic and aerobic gram-negative sepsis, although this finding must be balanced against the known limitations of case reports including publication bias ,54,55,56ical TZP . HoweverIn judging the appropriateness of any empirical antibiotic, it is important to balance toxicity against benefit. There are few published reports of TZP-related toxicity in septic patients when used as monotherapy, although this review only included reports in patients with clinically or microbiologically diagnosed sepsis. TZP appears appropriate for patients at risk of renal impairment, as a large retrospective cohort study of patients with gram-negative bacteraemia found no association of TZP receipt or duration of therapy with nephrotoxicity . PatientNeurotoxicity at high concentrations of penicillins, including piperacillin, is a known adverse effect and has been described in case reports , but theTZP-induced thrombocytopaenia was reported in two case studies ,41 and iP. aeruginosa bacteraemia, maintaining ≥60% of the dose interval above the MIC is a significant predictor of in-hospital survival [TZP may only be an appropriate empirical antibiotic for sepsis of unknown origin if dosed to maintain optimal therapeutic concentrations for sepsis due to organisms with high MIC. In survival . Despitesurvival . Howeversurvival  or serumsurvival . It is tExtended infusion regimens, where each dose is generally infused over 4 hours instead of 30 min, may improve TZP serum concentrations without the need for individualised TDM. An RCT in a Hong Kong ICU demonstrated significantly reduced 14-day mortality in bacteraemic patients receiving extended vs. standard intermittent TZP infusion . ConversThis review demonstrates the lack of studies directly addressing the research question. Therefore, the scope of the review was broadened to identify general themes that might be used to help frame the original research question. Nonetheless, some caveats apply to the proposed future use of these review findings in addressing the research question. Sepsis as an inclusion criterion was defined broadly as a clinical diagnosis of known or suspected sepsis, bacteraemia, or bloodstream infection or positive blood culture. Although a broad definition was needed to encompass all relevant research, patients receiving empirical TZP for sepsis of unknown origin will fulfil the clinical but not necessarily the microbiological definitions of sepsis used. Confining search results to patients with known infectious syndromes also limits knowledge of the effects of empirical TZP, particularly toxicity, on patients who have non-infectious causes for a sepsis-like presentation.Allowing for the lack of studies concerning sepsis of unknown origin, this review focused more broadly on sepsis of any source. A future review could be used to analyse only studies with a septic focus classified as ‘unknown’ or ‘primary’. However, many studies do not stratify the analyses of TZP efficacy by infection focus, and typically, the sample sizes of patients with an unknown infection focus are small. Furthermore, such a limited analysis does not allow for recognition that a septic focus is often identified after commencement of empirical antibiotics.This review only included studies with a major focus on clinical outcomes related to TZP therapy and may thus exclude studies with a non-clinical focus or where clinical outcomes are a minor aspect but still contribute valuable information, for example, in vitro susceptibility data for important sepsis-causing pathogens and pharmacokinetics in certain patient populations. As this review included studies investigating either empirical or definitive TZP in sepsis, the interpretation of its findings is limited when TZP is used in the critical early period before the causative organism and its antibiotic susceptibilities are known.Apart from four RCTs and one prospective observational study, all studies discussed in this review are retrospective and observational. Unlike RCTs, retrospective observational studies are only designed to determine association, not causation, and are limited by the presence of uncontrolled confounding variables that could influence clinical outcome independently of variables of interest such as TZP treatment. Most retrospective studies reviewed here attempted to adjust for known confounders with multivariate analysis and case–control matching. However, both these techniques do not adjust for undocumented confounders and multivariate analysis has limited power with small sample sizes. Moreover, retrospective studies are more vulnerable to some biases than RCTs, such as selection, performance, and measurement biases.The contemporary literature centres on the role of TZP in MDR sepsis compared to broader spectrum agents such as carbapenems. This focus not only biases studies towards TZP therapeutic failure, but it also fails to recognise the potential misuse of TZP in non-MDR sepsis, where, in Australia, a narrower spectrum of antimicrobials may be more appropriate. Further research is required to determine the appropriateness of TZP compared to narrower spectrum options in Australia, where sepsis of unknown origin may be caused by non-MDR organisms.P. aeruginosa and anaerobic sepsis. High TZP serum concentrations have been weakly linked to neurotoxicity in ICU patients, but better controlled studies are needed to determine the clinical impact of this link. So far, there are insufficient data to support the use of extended infusion regimens of TZP in sepsis, as is reflected in the Australian guidelines. Due to the difficulties of conducting large RCTs of empirical antibiotics, the majority of studies included in this review were retrospective with small sample sizes. Although the emerging evidence is compelling, this review highlights an urgent need to prospectively evaluate the appropriateness of antibiotic interventions in the Australian setting; until then, there remains insufficient equipoise to recommend a change to current clinical practice.In conclusion, the appropriateness of TZP as an empirical agent for sepsis of unknown origin in Australia is unclear. Further research is required, particularly where antimicrobial stewardship efforts could substitute TZP for narrower spectrum agents. Despite some limitations, the highest quality evidence rejects TZP as a carbapenem-sparing agent in gram-negative ESBL producers. Limited evidence suggests that TZP may be an acceptable treatment for AmpC producers, but the impact of AmpC expression levels and genotype on TZP appropriateness is so far unknown and requires further investigation. Only low-quality studies have investigated the appropriateness of TZP in susceptible


This mini-review focuses on cognitive impairment in iNPH. This symptom is one of the characteristic triad of symptoms in a condition long considered to be the only treatable dementia. We present an update on recent developments in clinical, neuropsychological, neuroimaging and biomarker aspects. Significant advances in our understanding have been made, notably regarding biomarkers, but iNPH remains a difficult diagnosis. Stronger evidence for permanent surgical treatment is emerging but selection for treatment remains challenging, particularly with regards to cognitive presentations. Encouragingly, there has been increasing interest in iNPH, but more research is required to better define the underlying pathology and delineate it from overlapping conditions, in order to inform best practise for the clinician managing the cognitively impaired patient. In the meantime, we strongly encourage a multidisciplinary approach and a structured service pathway to maximise patient benefit. Idiopathic Normal Pressure Hydrocephalus (iNPH) is a clinical syndrome  derived Cognitive impairment in iNPH is not universal but is frequently present. Insidious onset and more prevalent causes of cognitive impairment, such as vascular dementia (VaD) and Alzheimer's disease (AD), make early diagnosis of iNPH challenging. Significant cognitive impairment in the absence of an early, prominent and typical decline in mobility mandates consideration of other underlying causes given their impact on prognosis and therapeutic decision making.The typical cognitive profile of iNPH is “subcortical” with impaired attention, reduced psychomotor speed and inefficient memory .VaD results in a similar profile, making these two entities difficult to separate on clinical grounds . CerebroThe commonest cognitive profile of AD, that of progressive amnesia , may notTypical presentations of Lewy body disease (LBD) , progresLittle has been published on frontotemporal dementia (FTD) and iNPH. FTD should not cause differential diagnostic problems as difficulties with mobility occur late in the course of the disease. If present early, and typical for iNPH, co-pathology should be suspected. A case report of a C9orf 72 positive patient, with co-occurrence of typical features of FTD and iNPH, described post-shunt improvements in gait and executive tests, while the behavioural disorder remained unaffected .The differential diagnostic assessment of patients suspected of having iNPH, who have early and/or significant cognitive impairment, requires clinical expertise in cognitive and atypical movement disorders to delineate the presenting symptoms and signs. The assessment should conclude with a probabilistic diagnostic statement attributing the findings either to a single  morbidity or postulating co-morbidity. Neuroimaging, neuropsychology and CSF biomarkers all provide important diagnostic information, emphasising that the best approach to managing patients suspected of having iNPH with significant cognitive impairment is the protocol-driven multidisciplinary team assessment .The existing evidence of cognitive outcomes after shunting has recently been systematically reviewed. Improvement was found in 61% of patients . The autProbable iNPH patients who also have clinical features or biomarker evidence of CVD , AD 26)26), or LLong-term outcome studies of treated iNPH patients suggest that the numbers developing dementia are significantly greater than in the general population , 28. A lNeither aetiology nor pathogenesis of iNPH are well understood . The potExperience from AD, may serve as a model for future research see : cooperaSince the delineation of iNPH as a clinical syndrome, imaging has been needed to demonstrate ventriculomegaly and help evaluate differential diagnoses, including alternative or co-existing causes for cognitive impairment . Here, wIn the context of iNPH a widely studied pattern of CSF space morphology is the combination of (i) hydrocephalus (Evans index ≥0.3), (ii) high-convexity/midline tightness and (iii) Sylvian fissure enlargement. This “disproportionately enlarged subarachnoid-space hydrocephalus” (DESH) , 44 is fA number of recent studies have now investigated the presence of DESH in the wider population, where it is found in 1–7% and associated with poorer cognition –52. A stDiffusion tensor imaging (DTI) is an MRI technique which measures orientation-specific water diffusivity to interrogate brain micro-structure and characterise white matter tracts. White matter injury and dysfunction are proposed components of iNPH pathogenesis and DTI has demonstrated differences in white matter when compared to healthy controls, particularly the corticospinal tract  and corpWhilst DTI provides measures of structural brain connectivity, MRI techniques are also able to probe functional connectivity. Resting state functional MRI (rsfMRI) examines correlations in brain activity, identifying sets of brain regions that activate simultaneously in the absence of a specific cognitive task. One such set, known as the default mode network (DMN) , has beeMoreover, further studies suggest that the dysfunction seen in iNPH may involve multiple networks in addition to the DMN , 70 and in vivo MR imaging techniques have been explored  is the main component of the plaques found in AD, a frequent co-morbidity in iNPH patients that contributes to cognitive decline  now offer the ability to identify Aβ non-invasively (Recent work has highlighted the potential of PET imaging for the  decline , 88, 89. decline . The PETvasively –94. Multvasively , 95–98, Individuals with iNPH perform significantly worse than controls on various cognitive measures , 99–101.Some studies report post-tap test improvements using either cognitive screens  or more Cognitive screens, such as the MMSE , are comEven studies that control for practice effects show variable outcomes. Kambara et al.  showed tAnother reason for variability in cognitive outcomes may be the presence of alternative or co-morbid neurodegenerative diseases. In one study with a median follow up of 4.8 years, 80% of a shunt responsive group demonstrated cognitive decline, and 46% met the criteria for dementia, with the most common diagnoses being AD and VaD . The besIn studies examining neurodegenerative biomarkers, the results are also mixed. A higher incidence of AD biomarkers has been reported in iNPH compared with controls, and was associated with cognitive decline at 2 years . While iSpecific neuropsychological markers can aid in distinguishing iNPH from other neurodegenerative disorders , 125–127Cognitive impairments in iNPH typically involve executive dysfunction, but may be accompanied by more widespread deficits. Poorer cognition is associated with older age, longer disease duration, co-morbidity, variable outcomes after shunt surgery, and increased mortality.Variable outcomes may be due to inadequate control for confounding factors, inadequate cognitive measures, or whole-group analyses which average-out individual variability see . Where sRobust neuropsychological methods that control for practice effects in serial testing are needed . DetaileWe have presented evidence to inform patient management for practitioners confronted with cognitively impaired patients in whom a suspicion of iNPH has been raised. Recent developments have helped to improve differential diagnosis and patient selection for treatment. Neuropsychological differential diagnosis, advanced imaging, and CSF biomarkers are powerful tools starting to enter mainstream clinical use. We encourage active management of these patients through the optimal use of these tools within a structured clinical service. Hence the complex needs of patients with iNPH are best met within a multidisciplinary team. The nosology requires further clarification in prospective cohort studies in cooperation with basic science. An iNPH specific biomarker would revolutionise the field. However, agreement needs to be reached on standardised assessment methods and outcome measures of gait and cognition, where advanced neuropsychological batteries may serve to stratify clinical populations by cognitive features in future RCTs.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.