This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used. Although the global burden of helminth infections is still considerable ,9,10,11,Multiple genus- and species-specific PCRs and real-time PCRs ,26,27,28While superior sensitivity of real-time PCR for protozoan parasites compared to microscopy is considered to be well established , reliabiIn the present study, a standard nucleic acid extraction scheme was compared with a harsh, bead-beating-based extraction protocol and applying multiple different helminth real-time PCRs. This was done in order to assess whether and to what extent increased sensitivity after harsh extractions is a general or a species-dependent effect.The study was performed as a head-to-head comparison of two different nucleic acid extraction procedures regarding their effects on various diagnostic helminth real-time PCRs. Thereby, it was organized in a stepwise algorithm. In a first step, the test characteristics of the applied in-house helminth real-time PCRs were investigated. In a second step, the primary objective of the study, i.e., the comparative effects of nucleic acid extraction by either the QiaAMP DNA stool mini kit  or a harsher, bead beating-based nucleic extraction procedure, was assessed. In a third step, the effects of the harsh bead beating-based nucleic extraction procedure on real-time PCRs targeting nonhelminth pathogens like bacteria and protozoa, which are also of diagnostic interest for the assessment of human stool samples in the medical diagnostic laboratory, were analyzed in comparison to the QiaAMP DNA stool mini kit-based nucleic acid extraction scheme. This was done to exclude harmful effects of such a harsh approach on the quality of DNA of less robust nonhelminth pathogens. The different steps are visualized in Diagram 1 and described in the following.Diagram 1: Flowchart of the stepwise experimental approach.Specificity testing of the group-specific helminth real-time PCRs with helminth DNASensitivity and specificity testing of the group specific helminth real-time PCRs with DNA from stool samples positive in genus- and species-specific helminth real-time PCR after DNA extraction applying the QiaAMP DNA Stool Mini KitSensitivity and specificity testing of the in-house helminth real-time PCRs with DNA from stool samples positive for helminth eggs or larvae in microscopy after DNA extraction applying the QiaAMP DNA Stool Mini Kit1. Estimating the test characteristics of the applied in-house real-time PCRs:Historic residual stool samples which had been positive before in any helminth real-time PCR after nucleic acid extraction using the QiaAMP DNA Stool Mini KitStool samples from a Dutch external quality assessment scheme for helminths2. Comparing the effects of nucleic acid extraction applying the QiaAMP DNA Stool Mini Kit and a harsher bead beating-based nucleic extraction scheme with:Historic residual stool samples which had been positive before in any bacteria real-time PCR after nucleic acid extraction using the QiaAMP DNA Stool Mini KitHistoric residual stool samples which had been positive before in any protozoa real-time PCR after nucleic acid extraction using the QiaAMP DNA Stool Mini Kit3. Comparative assessment of the effects of nucleic acid extraction applying the QiaAMP DNA Stool Mini Kit and a harsher bead beating-based nucleic extraction scheme on nonhelminth pathogens which might also be relevant in the medical routine diagnostic laboratory with:Trichinella spiralis, Fasciola hepatica, Toxocara canis, Toxocara cati, Angyostrongylus vasorum, Dipylidium caninum, Schistosoma mansoni, and Strongyloides ratti, respectively, to exclude or confirm specificity limitations. Helminth tissue was provided from specimen collections from the institutions of the coauthors and the colleagues listed in the acknowledgements. Nucleic acid extraction was performed with the harsh bead beating-based procedure as described below.As a first step, all group specific helminth real-time PCRs as described below were assessed with helminth DNA of As a second step, both sensitivity and specificity of the group specific helminth real-time PCRs were analyzed using 96 residual DNA volumes from stool samples of patients with real-time PCR-confirmed enteric helminth infections. As negative controls, 100 residual nucleic acid extractions from stool samples of German citizens without history of travel and thus a very low likelihood of being infected with helminths were applied. Nucleic acids had been extracted using the QiaAMP DNA stool Mini Kit . The eluates had been stored at −80 °C prior to the assessment for time periods between few months and more than 10 years.Strongyloides infection. In fact, Strongyloides larvae had been seen after enrichment according to the Baermann method only. In detail, the 37 microscopically positive stool samples, i.e., samples in which at least one helminth egg or one helminth larva with potential pathogenic relevance for human patients had been microscopically observed, included had shown 29 nematode stages comprising 13 samples with Strongyloides stercoralis larvae; five with hookworm eggs; four with Ascaris lumbricoides eggs; three with Trichuris trichiura eggs; one with A. lumbricoides and T. trichiura eggs; as well as three with Enterobius vermicularis eggs; five samples with trematode eggs comprising one with small trematode eggs (suggestive of Clonorchis sinensis/Opisthorchis viverrini) and four with Schistosoma mansoni eggs; as well as three with Hymenolepis nana eggs as representatives of cestodes. Microscopical quantification had not been performed, i.e., the exact number of observed helminth eggs or helminth larvae per assessed microscopical slide had not been recorded. In the investigators’ experience, however, helminth loads in travel returnees are usually low.Prior to the nucleic acid extraction assessment comparisons, the applied in-house stool real-time PCRs for helminths as detailed below were tested with a small number of 37 nucleic acid extractions from microscopically positive stool samples from returning travelers with gastrointestinal symptoms after tropical journeys assessed at the Bernhard Nocht Institute for Tropical Medicine Hamburg, Germany. The samples had been collected within an eight-year interval (2006–2014) and extracted using the QiaAMP DNA stool mini kit (Qiagen). The eluates had then been stored at −80 °C. Native stool was no longer available from those samples, but the eluates were assessed in order to obtain an impression of baseline sensitivity of the applied real-time PCRs. Parasite microscopy that had been performed on the fresh stool samples was based on standard approaches including enrichment using sodium acetate–acetic acid–formalin solution (SAF) ,36,37 anAfter this, the study was conducted as a head-to-head comparison of nucleic acid extractions using 67 residual materials from stool samples that had shown positive real-time PCR signals for helminths in previous diagnostic assessments at the Bundeswehr Hospital Hamburg, Department for Microbiology and Hygiene, external site at the Bernhard Nocht Institute for Tropical Medicine Hamburg, Germany. Those stool samples had originally been obtained for diagnostic screening purposes from soldiers and policemen returning from tropical deployments, migrants travelling under poor hygiene conditions, and study participants from resource-poor tropical settings over the course of 13 years (2006–2019). No microscopic results were available for those assessed stool samples. Both bead beating-free and bead beating-based nucleic acid extractions as detailed below and residual stool samples had been stored at −80 °C prior to further assessment. Ethical clearance allowed only the anonymous use of residual sample materials for test comparison purposes in a fully anonymized way. Accordingly, no detailed patient-related information can be presented. In addition to the 67 diagnostic samples used, 10 stool samples from a Dutch external quality assessment scheme for helminths as described elsewhere  were incSalmonella spp., Shigella spp./enteroinvasive Escherichia coli (EIEC), Campylobacter jejuni, or Yersinia spp. [Entamoeba histolytica, Giardia duodenalis, Cryptosporidium spp., or Cyclospora spp. [To assess the effects of harsh bead-beating-based nucleic acid extraction on nonhelminth real-time PCR targets, the 67 residual stool samples with positive in-house real-time multiplex real-time PCR results for the enteroinvasive bacteria nia spp.  as well ora spp.  were sub−1, proteinase K digestion, and QiaAMP spin column extraction (Qiagen) as described previously [For the initial diagnostic assessment, nucleic acid from all samples had been extracted using the QiaAMP DNA Stool Mini Kit  as described by the manufacturer without particular focus on specialized procedures for parasite DNA (later referred to as “without bead beating”). If any helminth real-time PCR signal had been detectable in the initial diagnostic process and residual stool material was still available stored at −80 °C, nucleic acid from these residual materials were extracted using polyvinylpyrrolidone pretreatment, bead-beating using garnet beads  with a TissueLyser LT device (Qiagen) for 3 min at 30 seviously . This prAncylostoma spp., A. lumbricoides, E. vermicularis, H. nana, Necator americanus, Schistosoma spp., S. stercoralis, Taenia saginata, T. solium, and T. trichiura as well as commercial real-time PCRs  targeting Ancylostoma spp., A. lumbricoides, E. vermicularis, H. nana, N. americanus, Schistosoma spp., and S. stercoralis, as recently described [S. mansoni complex [S. haematobium complex [The total of 25 genus- and species-specific helminth real-time PCRs used comprised in-house real-time PCRs targeting escribed . Additio complex  and S. h complex , which w complex ,43 with  complex ,30 as siSamples were included if at least one positive pathogen-specific real-time PCR result had been observed during routine diagnostic assessment. There were no exclusion criteria, so samples were also included if residual sample material was insufficient for all assessments.Due to the low sample count of this proof-of-principle assessment, statistical approaches were basically descriptive. In detail, Cohen’s kappa was calculated to assess the agreement between the extraction schemes for each individual real-time PCR target. The interpretation standards as described  with theEthical clearance, provided for blinded use of residual materials for test comparison and evaluation purposes, was granted on 11 March 2019 by the ethics committee of the Medical Association of Hamburg, Germany (registration number WF-011/19) in line with national laws without requirement for informed consent. The authors assert that all procedures contributing to this work comply with the Helsinki Declaration of 1975, as revised in 2008.When applied with helminth DNA, individual cross-reactions of the group specific helminth real-time PCRs were seen. Such cross-reactions were associated with comparably high cycle threshold (Ct) values. Details of the Ct values of observed reactions and cross-reactions with nucleic acid extractions of helminths are shown in Applied with 96 residual DNA samples extracted from stool of patients with previous real-time PCR-based diagnosis of enteric helminth infections, imperfect sensitivity and specificity of the group specific helminth real-time PCRs was calculated. In detail, application of the group-specific real-time PCRs with samples positive in genus- or species specific helminth real-time PCRs (in-house protocols according to ) after Qn = 37), specificity of the in-house stool real-time PCRs for helminths after QiaAMP DNA Stool Mini Kit-extraction was generally good (96.7–100%). In contrast, there was a broad range of sensitivity ranging from 20.7% to 100% over the different in-house real-time PCRs for stool. Details of the assessments with the microscopically positive samples including cycle threshold values are provided in Schistosoma-specific blood real-time PCRs and the commercial assays. So, this quality control assessment had to be restricted to the real-time PCR assays as shown in As assessed using the nucleic acid residuals of the microscopically positive samples , while bead beating-based nucleic acid extraction (mean of 16.2 ng/µL) yielded about half as much DNA as nuclei acid extraction without bead beating (mean of 35.0 ng/µL) (p = 0.0003). p values were calculated using the Mann–Whitney U-test.After performing all real-time PCR assessments, sufficient residual sample volumes for photometric DNA quantification were available for 39 nucleic acid extractions without bead beating and for 77 nucleic acid extractions with bead beating as well as for 96 historic samples and 20 fresh external quality assessment scheme samples, respectively. There was no statistically significant difference between the DNA-concentrations in comparison of fresh external quality assessment scheme samples and historic samples (mean of 20.0 ng/µL for fresh quality control assessment scheme samples, mean of 23.0 ng/µL for historic samples, The study was performed to compare performance characteristics of two different nucleic extraction procedures for diagnostic real-time PCR approaches targeting helminths. A number of genus- and species-specific in-house helminth real-time PCR approaches ,42,43 suA. lumbricoides but also for fragile N. americanus eggs. For T. trichiura—a nematode with very robust eggs [As well as a standard approach for nucleic acid extraction from stool samples, a more sophisticated approach including bead-beating and proteinase K-digestion was applied as suggested in a recent publication . Most inust eggs —effects ust eggs ,34. NonsT. trichiura, for which the recorded Ct-values were similar. However, those differences are mostly lost when assessing the whole sample collection.Of note, however, considerable differences between real-time PCR results following the different extraction schemes suggest detections at the threshold of diagnostic sensitivity. Close to the detection threshold, positive results necessarily tend to become stochastic phenomena. There was a marked dominance of lower Ct-values after the bead-beating-based extraction scheme for the commercial test assays compared to the in-house assays. The reasons for this remain unclear: different sensitivity of the assays might theoretically play a role here. However, a previous assessment  did not T. trichiura, which is in contrast to previous results [Interestingly, however, not even the commercial assay showed unambiguous difference for the robust eggs of  results ,34. As d results , minor dShigella spp./EIEC after the bead-based nucleic acid extraction strategy was counteracted by only slight agreement, so this result is difficult in interpret. Significant differences in the measured Ct-values were never observed, however.Considerable, target-dependent, variability in agreement of the results following the different nucleic acid extraction strategies was observed for the real-time PCRs with nonhelminth targets. Significantly more detection of The present study has a number of limitations. First, the samples that were used for the extraction comparison were not assessed microscopically. So, definite microscopic proof of the presence of pathogens was not feasible. Secondly, due to the rarity of occurrence of helminth-positive sample materials, the overall numbers of samples analyzed was low. Thirdly, and for the same reasons, the ages of the samples used varied between a few months and about 10 years, although frozen storage at −80 °C makes quantitatively relevant nucleic acid degradation less likely. Fourthly, there was no homogenization step of the initially provided stool samples, making uneven distributions of parasites in stool likely. Fifthly, real-time PCRs after different extractions were not consistently performed in parallel in the same run, and low volumes of residual sample materials did not allow repeated testing. So, the comparison indeed reflects routine-like conditions. Sixthly, due to limited available sample volumes, photometric assessments of the DNA concentration within the eluates could be performed only for a subset of the samples included in the study. Accordingly, effects to varying DNA concentrations after the different extraction schemes could not be analyzed for all samples. However, an exploratory investigation with the available residual sample materials did not indicate significant influence of sample age on measured DNA concentrations. Although the extraction scheme without bead beating yielded about twice as much DNA compared to the extraction scheme with bead beating, an average factor of two will only have minor influence on measured Ct values in real-time PCR. Seventhly, individual fecal samples contain variable concentrations of inhibitory components , potentiIn spite of these limitations, the assessment indicated that the harsh nucleic acid extraction scheme could indeed provide considerable shifts in Ct-values for several samples and also provided tendencies of higher sensitivity. However, the observation of Ct-value shifts is inconsistent. Especially with historic sample materials, which may play a role in retrospective epidemiological assessments rather than for routine diagnostic applications, the beneficial effects of the harsh extraction approach are less obvious than in previous reports assessing fresh samples. Nevertheless, early international external quality assessment trials  have sugThis nucleic acid extraction comparison indicated inconsistent effects of the analyzed harsh nucleic acid extraction scheme on helminth real-time PCRs. There were only tendencies for beneficial effects of bead-beating-based extraction as observed with the samples from the external quality assessment scheme. If complex nucleic acid extraction schemes are infeasible for logistical reasons in diagnostic or study settings, sensitivity constraints may result. With historical samples in retrospective assessments, any beneficial effects of harsh nucleic acid extraction may be less pronounced than observed with fresh sample materials in the diagnostic routine in previous studies.


Flavonoids derived from citrus plants are favored by phytomedicinal researchers due to their wide range of biological activities, and relevant studies have been sustained for 67 years (since the first paper published in 1955). In terms of a scientometric and critical review, the scientometrics of related papers, chemical structures, and pharmacological action of citrus flavonoids were comprehensively summarized. The modern pharmacological effects of citrus flavonoids are primarily focused on their anticancer activities , neuroprotective effects , and metabolic diseases. Furthermore, the therapeutic mechanism of cancers , neuroprotective effects (including antioxidant and anti-inflammatory), and metabolic diseases  were summarized and discussed. We anticipate that this review could provide an essential reference for anti-cancer and neuroprotective research of citrus flavonoids and provide researchers with a comprehensive understanding of citrus flavonoids. C. reticulata), grapefruit (C. maxima), and citron (C. medica) . . 79]. Naringenin could also promote deoxyribonucleic acid (DNA) repair and prevent carcinogenesis caused by oxidative damage .Hesperetin exhibited potential anticancer activity in vitro against human cervical cancer cell lines by reducing cell viability and inducing apoptosis . Naringin also induced growth inhibition and apoptosis in human cervical cancer HeLa cell lines by activating the nuclear factor kappa-B (NF-κB)/Cyclooxygenase-2 (COX-2) -caspase-1 pathway .Nobiletin inhibited ovarian cancer cells by secreting key angiogenic mediators such as AKT, HIF-1α, NF-κB, and vascular endothelial growth factor (VEGF)  83]. . 83]. Tangeretin sensitized cisplatin-resistant human ovarian cancer cells by downregulating the PI3K/AKT signaling pathway  84]..84].Studies have shown that citrus flavonoids have anti-proliferative effects in inhibiting human squamous cell carcinoma in vitro , and theStudies have shown that fruits rich in flavonoids could protect the nervous system . Citrus Nobiletin could stimulate protein kinase A (PKA)-mediated phosphorylation of glutamate receptor 1 (GluR1) receptors in the hippocampus to upregulate synaptic propagation through postsynaptic AMPA receptors . It coulNaringin protected nigrostriatal nigrothymic dopaminergic (DA) projections from 6-hydroxydopamine (6-OHDA)-induced neurotoxicity . NaringiNaringenin exerted anti-inflammatory effects due to its interaction with the p38 signaling cascade and signal transducer and activator of the transcription 1 (STAT-1) transcription factor . NaringeHesperidin protected against cognitive impairment by inhibiting the overexpression of inflammatory markers such as NF-κB, nitric oxide synthase (NOS), and cyclooxygenase-2 (COX-2) . FurtherHesperetin attenuated lipopolysaccharide (LPS)-induced neuroinflammation, apoptosis, and memory impairment by regulating the toll-like receptor 4 (TLR4)/NF-κB signaling pathway , also coTangeretin significantly protected striatum–substantia nigra integrity  by inhibCitrus flavonoids protect the nervous system by fighting against inflammation and protecting the function of nerve cells. It is promising to develop citrus flavonoids as an adjuvant treatment for neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, etc. It is believed that the protective effects of citrus flavonoids on the nervous system is worthy of further and in-depth research.Oral administration of bergamot extract  for 6 months reduced moderate hypercholesterolemia, low-density lipoprotein, and blood lipids in patients with atherosclerosis . NeohespNobiletin attenuated dyslipidemia by preventing hepatic triglyceride (TG) accumulation, reducing very low-density lipoprotein (VLDL) and TG secretion , while iNaringenin reduced the progression of atherosclerosis by improving dyslipidemia , apolipoNaringin, the major grapefruit flavonoid, primarily affected the development of atherosclerosis in diet-induced hypercholesterolemia in mice  by modulDietary hesperidin exerted hypoglycemic and hypolipidemic effects in streptozotocin-induced borderline type 1 diabetic rats . Polymethoxyflavonoids are a novel flavonoid with cholesterol and triacylglycerol-lowering potential, and elevated levels of polymethoxyflavonoid metabolites in the liver may directly lead to its hypolipidemic effect in vivo . HesperiHesperetin alleviated hyperglycemia and dyslipidemia by improving the antioxidant capacity of streptozotocin (STZ)-induced experimental rats . HespereAfter oral administration of bergamot extract (BPF) to rats and patients for 30 days, BPF significantly reduced triglyceride levels and blood glucose. Meanwhile, BPF inhibited 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity and enhanced reactive vasodilation .After diosmin was administered orally (100 mg/kg/day) for 45 days, both histological and biochemical parameters demonstrated antidiabetic effects in type 2 diabetic rats .Based on the above-provided summary, the effect of citrus flavonoids on blood lipids was confirmed. We believe that further research on citrus flavonoids in metabolic diseases is valuable.In conclusion, digesting more citrus fruits in our daily diet is likely to be beneficial to our health. Additionally, Citrus flavonoids play a certain role in anti-cancer effects, neuroprotection, and metabolic regulation. Among the many citrus flavonoids, nobiletin is a promising anti-angiogenic agent with great potential for cancer prevention and treatment. In terms of neuroprotection, nobiletin could improve learning and memory deficits, suggesting its anti-Alzheimer’s disease and Parkinson’s disease potential. Moreover, hesperidin and naringin could prevent Parkinson’s disease-related dopaminergic neuron (DA) degeneration, oxidative damage, and cognitive impairment. In short, citrus flavonoids possess high nutritional and medicinal value.The production of citrus fruits is abundant around the world, and there is still the problem of insufficient utilization of citrus fruits in juice. Therefore, it is important to study and develop the residues of the industrial production of citrus juice. The existing underutilization problem can in turn provide supplements for human health. Moreover, systematic and in-depth research on the neuroprotective effect of citrus flavonoids should be carried out to comprehensively, deeply, and accurately evaluate the neuroprotective effect of citrus flavonoids. The nutritional value of citrus plants as food is also worth of attention, and the content of citrus flavonoids may be used as an indicator to evaluate the nutritional value. Finally, the effects of the long-term use of citrus flavonoids on our health are worthy of further investigation.


Graphene oxide (GO) is an oxidized form of graphene accommodating various oxygen-containing functional groups such as hydroxyl, epoxy, and carboxyl groups on its surface. GO has been extensively utilized in various biomedical applications including the delivery of biomolecules and the development of biosensors owing to its beneficial properties such as high surface area, nucleic acid adsorption, and fluorescence quenching through fluorescence resonance energy transfer (FRET). However, despite these favorable properties, the direct utilization of GO in these applications is often limited by low dispersibility in a physiological medium, cytotoxicity, low biocompatibility, and a strong binding affinity of nucleic acids to GO surface. The large surface area of GO and the presence of various functional groups on its surface make it highly amenable to facile surface modifications, offering scope for GO surface functionalization to overcome these limitations. When polyethylene glycol (PEG), which is a biocompatible polymer, is conjugated to GO, the PEGylated GO enhances the biocompatibility and dispersibility, reduces cytotoxicity, and allows controlled drug delivery with controllable binding affinity towards nucleic acid. PEG-engrafted GO retains the beneficial properties of GO while effectively addressing its limitations, rendering it suitable for various biomedical applications. In this review, we present the recent advancements of PEGylated GO in gene/drug delivery and the facilitation of nucleic acid amplification techniques, which aid in the development of therapeutic and diagnostic tools, respectively. Th. Th54]. Zhang et al. focused on the development of PEGylated reduced GO (PEG-RGO) as a potential delivery system for ssRNA molecules . The res® QIAGEN Inc. . Furthermore, functional gene delivery was confirmed through c-Myc protein knockdown and EGFP gene expression, underscoring the potential of this nanocarrier. The study’s significant contributions lie in its dual functionality and efficient nucleic acid delivery capabilities. The authors acknowledge the need for further improvement, particularly in enhancing targetability and endosomal escape, which are crucial for clinical applications.Imani et al. presented a comprehensive investigation of a dual-functionalized GO-based nanocarrier with the conjugation of aminated-polyethylene glycol (PEG-diamine) and octa-arginine (R8) for the intracellular delivery of nucleic acids in the treatment of cancer . This stYin et al. investigated the application of PEGylated GO nanosheets for combined photothermal and gene therapy in the context of pancreatic cancer . As showThe study conducted by Feng et al. explored the development of a novel nanomedicine platform by GO with PEG and polyethylenimine (PEI) to create a dual-polymer-functionalized nano-GO conjugate, referred to as NGO-PEG-PEI . This hyA study by Yadav et al. explored the covalent tethering of poly(amidoamine) (PAMAM) and PEG-modified GO to create a hybrid vector known as GPD , in whicThe study conducted by Szénási et al. expanded the previous findings that superoxide dismutase 1 (SOD1) overexpression in ovarian cancer leads to platinum resistance due to its role in conferring oxidative stress resistance against platinum compounds . PlatinuPEGylated GO enhances the pharmacokinetics of drug delivery systems due to its high loading capacity and low toxicity. The attachment of PEG chains increases the circulation time of GO in the bloodstream, effectively extending the duration during which it can accumulate at target tissues. This leads to a higher concentration of drug delivery at the desired sites, significantly improving the efficacy of therapeutic treatments.PEGylated nano GO (NGO-PEG) has been utilized by Lie et al. for delivering water-insoluble anticancer drugs into human colon cancer cells . The stuThe study by Shen et al. functionalized GO with an amine-terminated 6-armed PEG , enhanciThe study by Xu et al. discussed the use of PEGylated GO as a nanocarrier for delivering the chemotherapy drug Paclitaxel (PTX)  47]. PT. PT47]. Miao et al. investigated the safety and potential of PEG-grafted GO (pGO) nanosheets as a versatile nanocarrier for the co-delivery of photosensitizers and anticancer agents . Synthes10K-6arm) for the delivery of hydrophobic anticancer drugs, specifically oridonin and methotrexate (MTX)  a 59]. G. G10K-6aIn another study carried out by Dembereldorj et al., the authors explored a novel spatiotemporal anticancer drug release platform involving PEGylated GO (PEG-GO) and its response to glutathione (GSH) triggers b 71]. T. T71]. TA study by Pei et al. explored the use of PEGylated nano-GO (pGO) as a nanocarrier for delivering a combination of anticancer drugs, cisplatin (Pt) and doxorubicin (DOX), to improve anticancer activity . In thisThe study performed by Wen et al. focused on addressing a significant challenge in the field of intracellular drug delivery using PEG-functionalized nano-graphene oxide (NGO) . PEGylatAnother study focused on the development of (PEG) decorated graphene oxide GO nanosheets for controlled release of curcumin, an anti-cancer drug . GO was In another study, a PEG-bis amine (PEGA) functionalized GO/iron oxide nanocomposite was synthesized to serve as a drug-loading platform, with methotrexate (MTX) as the model anticancer drug . CytotoxYao et al. presented a comprehensive investigation into the development of a multifunctional GO drug carrier for the targeted delivery and controlled release of Dox into cancer cells, particularly hepatocarcinoma cells . The synA nanocarrier was synthesized by amidation, grafting folate-terminated PEG (FA–PEG–NH2) onto carboxylated GO nanosheets (CG) . In termJeshvaghani et al. introduced a pH-responsive, sustained-release nanocomposite composed of PEG, GO, and natural silk fibroin (SF) protein . Using aYang et al. demonstrated loading of FAM-cDNA21 and Dox onto the GO surface, followed by their efficient uptake by cancer cells . In the A study conducted by Yu et al. addressed the challenge of treating depression, a chronic mental disorder with significant health implications, largely due to the restrictive nature of the blood–brain barrier (BBB) . This baA recent study by Demirel et al. showed efficient delivery of Dox and anti-GAPDH siRNA via PEG-conjugated reduced GO. An azide functionality containing poly[(poly(ethylene glycol) methyl ether methacrylate)-co-(3-azidopropyl methacrylate)-co-(methyl methacrylate)-co-(1-pyrenemethyl methacrylate)] (P(PEGMA-co-AzPMA-co-MMA-co-PMA), PAMP) copolymer was synthesized, and this copolymer was conjugated on the surface of rGO (PAMP-CP-rGO) for controlled release of dual-loaded nanocomposite. This dual-loaded nanocomposite showed high transfection efficiency and selective toxicity against cancer cells only, making it a safe and adaptable delivery platform .50 values of 7 and 5 mg/mL, respectively. Notably, these values closely align with the IC50 observed for pure PTOX [Islam et al. synthesized a nanodrug delivery system by loading podophyllotoxin onto a GO-PEG surface (GO/PEG/PTOX) with a 25% loading ratio and 50% drug release after 48 h. It was demonstrated that GO/PEG/PTOX nanocomposite can be applicable in type-2 diabetes mellitus treatment as it efficiently inhibited α-amylase and α-glucosidase, exhibiting ICure PTOX .Hu et al. synthesized a nanocomposite where PEG-GO was conjugated with lectin protein-Con A and pesticide epoxiconazole (EPX) to increase the antifungal activity of EPX against spore germination in Magnaporthe oryzae. This GO-PEG-ConA nanocomposite inhibited the spore germination in M. oryzae by 41.7% suggesting its potential role in controlling rice blast lesions in rice seedlings .Nucleic acid amplification technologies (NAATs) such as polymerase chain reaction (PCR), quantitative real time PCR (qPCR), and loop-mediated isothermal amplification (LAMP) have revolutionized molecular diagnostics by enabling easy, sensitive, specific, and high-throughput detection of a wide range of infectious diseases ,81. Our 2+) and reduces the non-specific binding of Taq DNA polymerase onto the GO surface, allowing unhampered activity of Taq DNA polymerase [PCR is a synthetic amplification technique that offers extensive utility in diagnostics and molecular biology ,83 but ilymerase . It was PCR amplification of the target gene was carried out in the presence of three carbon composites , and PEG-nGO) and interestingly, a specific PCR-amplified band was only observed in the presence of PEG-nGO. This intriguing observation prompted us to investigate the affinity of PCR components such as ssDNA and Taq DNA polymerase with these three carbon composites. GO and nGO exhibited stronger binding affinities towards single-stranded DNA than PEG-nGO, which indicated that the weaker binding affinity of PEG-nGO towards single-stranded DNA is more suitable for PCR reactions. Furthermore, Taq DNA polymerase was found to bind to GO and nGO, leading to inhibited enzymatic activity, whereas PEG-nGO did not bind to Taq DNA polymerase, preserving its enzymatic activity. We evaluated the impact of PEG-nGO on multiple rounds of PCR since they are prone to produce non-specific amplicons due to the higher concentration of amplified DNAs. In the consecutive rounds of PCR, only the target DNA bands were observed in the presence of PEG-nGO without any smearing, whereas nonspecific smeared bands were observed in the absence of PEG-nGO. Next, to assess the effect of PEG-nGO on primer dimer formation, primers were added to the PCR mixture without any template. In the absence of PEG-nGO, bands indicating dimerized primers were observed, but these dimerized primer bands disappeared when PEG-nGO was added to the PCR mixture. PEG-nGO also demonstrated reduced mispairing between template DNA and primers at low annealing temperatures by adsorbing excess primers on its surface, thereby enhancing the specificity of PCR. This study demonstrated that PEG-nGO significantly enhanced the specificity and efficiency of PCR by adsorbing excess primers in the initial stage of PCR, which resulted in reduced primer dimerization and prevented reannealing of amplified DNA in later stages of PCR, facilitating primer annealing to template strands.Encouraged by the beneficial effect of PEG-nGO on end-point PCR assay, our group expanded the applicability of PEG-nGO by investigating the effect of PEG-nGO on quantitative fluorescence-based qPCR assay . qPCR isTo explore the hypothesis that PEG-nGO can enhance the specificity of qPCR by adsorbing excess primers in the initial phase of qPCR, thus reducing primer dimerization and false priming, we utilized dsDNA dye-based qPCR to detect influenza viral RNA as a model system a. We comLoop-mediated isothermal amplification (LAMP) is a rapid and robust NAAT which is carried out at isothermal temperature, thus eliminating the need for a thermocycler ,95,96. LMotivated by PEG-nGO-based enhanced specificity in PCR, we investigated its potential in LAMP, aiming to exploit its ssDNA adsorption abilities with multiple ssDNA primers . We testGO has firmly established itself as a revolutionary material with significant implications in the field of biomedicine. Its distinctive characteristics, such as its large surface area, facile surface modification, hydrophilicity, moderate electrical conductivity, robust mechanical properties, and ability to bind with a variety of biomolecules have proven greatly beneficial in advancing drug delivery systems and biosensors. Despite having all these advantageous properties, the utilization of GO is often limited to various biomedical applications. These limitations include poor solubility, cytotoxicity, generation of immune responses, nonspecific binding of various biomolecules on its surface, and strong binding affinity to nucleic acids. To overcome these limitations, GO can be easily conjugated with biocompatible polymer PEG, due to its larger surface area and presence of various oxygen containing functional groups.PEGylation of GO offers several advantages such as imparting adequate solubility and stability in various physiological media, minimized toxicity, specific binding of biomolecules, enhanced retention time of drugs, and weaker binding affinity of nucleic acid on its surface. Thus, PEG-conjugated GO not only retains the beneficial properties of GO but also effectively addresses its limitations, positioning it as a favorable option for a wide array of biomedical applications. Therefore, PEGylated GO has the potential to aid the development of therapeutic solutions in the biomedical field.


TERT, that steadily increased after initial HCC diagnosis till recurrence. One LC examined had HBV-TERT DNA detected, but transiently in 3.5 years during HCC surveillance. HBV-JS DNA was persistently elevated prior to the diagnosis of recurrent HCC, suggesting the potential of urinary HBV-JS DNA to detect MRD and HCC recurrence after treatment.Hepatitis B virus (HBV)-host junction sequences (HBV-JSs) has been detected in the urine of patients with HBV infection. This study evaluated HBV-JSs as a marker of minimum residual disease (MRD) and tumor recurrence after treatment in HBV-hepatocellular carcinoma (HCC) patients. Archived serial urine DNA from two HBV–HCC with recurrence as confirmed by MRI and four HBV-related cirrhosis (LC) patients were used. Urinary HBV-JSs were identified by an HBV-targeted NGS assay. Quantitative junction-specific PCR assays were developed to investigate dynamic changes of the most abundant urinary HBV-JS. Abundant urinary HBV-JSs were identified in two cases of tumor recurrence. In case 1, a 78-year-old female with HBV- HCC underwent a follow-up MRI following microwave ablation. While MRI results were variable, the unique HBV-JS DNA, HBV-Chr17, steadily increased from initial diagnosis to HCC recurrence. In case 2, a 74-year-old male with HBV–HCC contained two HBV-JS DNA, HBV-Chr11 and HBV- Liver cancer is the sixth most commonly diagnosed cancer and the third most common cause of cancer death worldwide, with an estimated 900,000 new liver cancer cases and 830,000 deaths during 2020 . HepatocEarly Detection of MRD is important for disease management to improve prognosis. Currently, there are no useful postoperative recurrence markers or specific guidelines for how HCC recurrence should be monitored. It has been difficult to detect recurrent HCC and MRD using alpha-fetoprotein (AFP) and desgama–carboxy prothrombin (DCP) measurements which have limited sensitivity and specificity in patients with non-tumorous hepatic disorders and post-ablation due to active hepatic necrosis that can result in increased levels  and by sCell-free tumor DNA (ctDNA), the DNA derived from apoptotic tumor cells released into circulation from tumors, is a promising tumor marker with versatile applications, including monitoring tumor progression or resistance to chemotherapies ,21,22,23Hepatitis B virus (HBV) is the leading cause of liver cancer, contributing to 60% of HCC in Africa and East Asia. Integrated HBV DNA has been shown to be in 80–90% of HBV-related HCC ,33,34,35To evaluate the urinary HBV-JS DNA as a potential indicator of minimum residual disease (MRD) and for monitoring tumor recurrence in patients with HBV–HCC, we first identified HBV–HCC cases that met the following criteria for investigation: (1) having HCC recurrence confirmed by MRI, (2) with at least four available serial archived urine cell-free DNA (ucfDNA) with one serial DNA sample containing at least 20 ng DNA that could be used for HBV-targeted NGS assay for HBV-JS detection as previously described , and (3)A 78-year-old female with HBV-related cirrhosis was diagnosed with HCC in 2015. After microwave ablation, the patient was monitored for recurrence by MRI and serum AFP. Follow-up MRI revealed a new LI-RADS 3 lesion 1 year later with serum AFP of 2.65 ng/mL. Subsequent imaging remained radiographically indeterminate until 2018, when the lesion was classified as definite HCC (LI-RADS 5).The last available archived urine DNA for HBV-JS NGS analysis was collected over 2 years (8/2016) before the MRI diagnosis of recurrence which was in 2018. We detected one HBV-JS integrated in Chromosome 17 non-coding region (HBV-Chr17) with ≥2 SR . To asseInterestingly, while serial AFP levels were negligible and MRI results variable, the unique HBV-JS DNA, HBV-Chr17, steadily increased from initial diagnosis of 1.2% in 2015 to 3.0% and then 7.0% in August 2016, prior to diagnosis of HCC recurrence in October 2018. There was no urine collected when HCC was diagnosed. HBV-Chr17 was detected right after treatment and steadily elevated, suggesting the presence of MRD and suspicion of HCC recurrence from the same clone that contained HBV-Chr17. The patient passed away shortly.A 74-year-old male with HBV-related cirrhosis was diagnosed with HCC in 2014, which recurred with an LI-RADS 5 lesion after 1 year despite loco-regional therapy.TERT gene (HBV-TERT_1) with 22 SRs, and GCSHP2 (HBV-GCSHP2) with 3 SRs as listed in TERT integration (HBV-TERT_2) with 2 SRs. Note this less abundant HBV-TERT_2 contains a different host-viral integration breakpoint from HBV-TERT_1.The last available archived ucfDNA used for HBV-JS detection was 3 months before recurrence diagnosis by MRI. Four HBV-JS with ≥2 SRs were detected from the urine specimen collected on 4/2015. Of which, the three most abundant HBV-JSs are integrations in non-coding sequences in Chromosome 11 (HBV-Chr11) with 63 SRs, TERT) detailed in Our hypothesis is that the tumor-derived viral-host junction DNA should be the most abundant among HBV-JSs detected in urine because of clonal expansion in tumorigenesis. Interestingly, HBV-Chr11 and HBV-TERT_1 junctions are much more abundant than HBV-GCSHP2, suggesting HBV-Chr11 and HBV-TERT_1 junctions might be HCC-derived. We thus selected HBV-Chr11 and HBV-TERT_1 for subsequent study of biomarker dynamics in serially collected urine, with clinical time points and HCC treatment detailed in As shown in TERT junction was identified with 9 SRs that were identical to HBV-TERT_1 found in case 2 , HBV-JS DNA markers were elevated before and at the time of diagnosis by MRI imaging. MRI/CT imaging is the gold standard for diagnosing recurrent HCC, but its ability to detect early recurrence, especially after local ablation in the previously treated areas, is limited. This may explain why the DNA markers were found in urine earlier than the MRI diagnosis. Secondly, HCC, like other solid cancers, is a disease of the genome. Detection of HCC genetics should provide not only sensitive and earlier detection for monitoring HCC recurrence and unambiguous assessment for MRD and may also provide HCC genetic information to assist in patient management. Integrated HBV DNA, found in >85% of HBV–HCC, can play a significant role in HBV-related liver disease progression. An HBV-JS created through each integration event provides a molecular signature for each infected hepatocyte and has been used to determine HBV–HCC clonality. Its high abundance can be used as a biomarker for clonal expansion.TERT junctions, the most frequent recurrently targeted gene by HBV integration found in HCC patients [TERT junction coordinates should be unique as HBV integration is through a non-sequence-specific mechanism. However, in our previous study, we found that 76% of TERT integrations (n = 477) mainly occur in a small 15 kb region upstream of exon 1, and within that region, over 50% were found in the ~1kb promoter region . This small window of frequent TERT integration contained 10% of HBV-TERT integrations that shared identical viral-host breakpoints. It may not be surprising that the HBV-TERT junction identified in case 2 and case 3 of this study coincided with the previously reported repeat HBV-TERT integration.Interestingly, in this pilot study of six patients, we detected two identical HBV-patients . The majTERT, for case 3 LC, it was only detected transiently. This could be due to the interplay of the host immune system, tumorigenesis, and the existence of HBV DNA integration events, as we have previously suggested [TERT integration was detected in urine from an LC patient (case 3). It is possible that the TERT integration was derived from either neo-antigen expression or indicative of tumorigenesis. Likely, its elimination by the host immune system resulted in a spike of detectable HBV-TERT junction in urine. On the other hand, detection of persistently elevated HBV-JS marker values reflects persistent tumor growth whereby some tumor cells that harbored HCC-derived junctions were eliminated by apoptosis, thus serving as an important biomarker for HCC MRD and recurrence monitoring, as schematically shown in As we’ve demonstrated previously  that HBVuggested . Here, nAlthough this study is limited by the small cohort size, it provides feasibility evidence that detection of highly abundant HBV-JS in urine from patients with HBV–HCC  could be HCC-derived and served as an HCC marker for monitoring HCC recurrence and MRD assessment in a noninvasive urine liquid biopsy. Furthermore, since the collection of urine can be done at home and then shipped to certified laboratories for testing, the urine test can result in better compliance without the need for a doctor’s office visit.It is important to note that the levels of urine DNA markers fluctuate for several reasons, including the hydration of the patient at the time of collection, which can result in diluted DNA in the urine. Further studies are needed to identify a proper internal control for this work, which is currently in progress.Despite the lack of FDA-approved HCC therapies since Sorafenib, the first FDA-approved HCC chemotherapy over 10 years ago, the recent approval of the immune checkpoint inhibitors (ICIs) PD-L1 inhibitor atezolizumab plus the antiangiogenic agent bevacizumab as front-line treatment for advanced HCC and the IMbrave 150 phase III trial has established a novel standard of care in a relatively short period of time. Despite this unprecedented treatment paradigm shift in HCC treatment, only a proportion of HCC patients benefit from immunotherapy, highlighting the need for biomarkers for assessing MRD and predicting treatment response. Encouragingly, the advent of molecular profiling has produced notable promise in the management of HCC associated with response to ICIs. Genomic aberrations such as Wnt/β-catenin mutations in HCCs have been reported to be significantly associated with resistance to immunotherapy compared to wild-type patients, emerging as potential predictive biomarkers of immunotherapy. The HCC medical community is called to put more effort aimed at evaluating novel biomarkers of response to ICIs, considering tumor-intrinsic , microsatellite instability (MSI) status, etc.), immune-specific, and combinatorial biomarkers ,44,45. TIn conclusion, we have demonstrated the potential of urinary HBV-JS DNA for the early detection of HCC recurrence and MRD assessment. Furthermore, we concluded that marker dynamics, such as persistent elevations of a highly abundant HBV-JS DNA, are important for the clinical relevance of HCC detection.


Pt-Mal-LHRH is a newly synthesized chemotherapeutic agent that was designed to selectively target the luteinizing hormone-releasing hormone (LHRH) receptor expressed by triple negative breast cancer (TNBC). The aim of this study was to evaluate the therapeutic dosing, tumor reduction efficacy, and selective distribution of Pt-Mal-LHRH in-vivo.LHRH tissue expression levels in-vivo were investigated using western blotting and LHRH was found to be increased in reproductive tissues . Further, Pt-Mal-LHRH was found to have increased TNBC tumor tissue platinum accumulation compared to carboplatin by inductively coupled plasma mass spectrometry analysis. The platinum family, compound carboplatin, was selected for comparison due to its similar chemical structure and molar equivalent doses were evaluated. Moreover, in-vivo distribution data indicated selective targeting of Pt-Mal-LHRH by enhanced reproductive tissue accumulation compared to carboplatin. Further, TNBC tumor growth was found to be significantly attenuated by Pt-Mal-LHRH compared to carboplatin in both the 4T1 and MDA-MB-231 tumor models. There was a significant reduction in tumor volume in the 4T1 tumor across Pt-Mal-LHRH doses (2.5–20 mg/kg/wk) and in the MDA-MB-231 tumor at the dose of 10 mg/kg/wk in models conducted by an independent contract testing laboratory.Our data indicates Pt-Mal-LHRH is a targeting chemotherapeutic agent towards the LHRH receptor and reduces TNBC tumor growth in-vivo. This study supports drug conjugation design models using the LHRH hormone for chemotherapeutic delivery as Pt-Mal-LHRH was found to be a more selective and efficacious than carboplatin. Further examination of Pt-Mal-LHRH is warranted for its clinical use in TNBCs, along with, other reproductive cancers overexpressing the LHRH receptor. Identification of new chemotherapeutic compounds that target triple negative breast cancer (TNBC) is critical for enhancing patient outcomes and survival rates. About 10–20% of all breast cancers are considered TNBC, in which, cells lack the expression of the estrogen, progesterone, and human epidermal growth factor receptor 2 (HER2/neu) proteins . TNBC is50) and smaller therapeutic window than carboplatin; which requires monitoring of a lower dosing regimen due to the increased toxicity [The platinum family of chemotherapeutics have been a predominant treatment option for patients afflicted with reproductive cancers including breast. Platinum compounds are a treatment option for TNBC including the use of carboplatin as a neoadjuvant chemotherapy regimen . Platinutoxicity . Due to toxicity , 8. Substoxicity , 10. To One such approach is to overpower resistance mechanisms by increasing intracellular concentration of the drug by targeting cancer cell-specific receptors. Luteinizing hormone-releasing hormone (LHRH), also referred to as gonadotropin releasing hormone, is a decapeptide regulatory hormone involved in reproduction and receptors are found on reproductive tissues , 12. NumVarious therapeutic modalities have used LHRH conjugation for a targeting approach including curcumin-LHRH, gemcitabine-LHRH, and lytic peptide-LHRH , 20. Fur2 and passaged until the desired number of cells was reached.The mouse tumor cell line 4T1 was purchased from American Type Culture Collection (ATCC). The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) along with 10% Fetal Bovine Serum, 100 U/mL penicillin and 100 μg/mL streptomycin. They were cultured in a 37ºC tissue culture incubator at 95% humidity and 5% CO6) were suspended in 100uL of DMEM not supplemented with FBS and injected into the right mammary fat pad. After orthotopic tumor initiation (~100mm3) the mice were allocated into treatment groups based on equal tumor volumes and treated with Pt-Mal-LHRH  by intravenous injection as day 1 and tumor growth was monitored for 7 days after. All treatment doses and volume measurements were conducted for each group at the same time of day during each assessment period. The tumor volumes (mm3) were calculated using the formula (width)2 X length/2, where the length is the smaller of the two measurements. End tumor volume was calculated, and blood samples were collected by left ventricular puncture. Blood was mixed with EDTA and cell blood count data was collected by Hemavet analysis.Female Balb/c mice, 10wk old were purchased from Jackson Laboratory  and housed as previously described by Calderon, et al., 2017 . 4T1 celUsing the mouse breast cancer model described above, mice were treated with carboplatin or Pt-Mal-LHRH by intravenous injection and tumor growth was monitored for two weeks. Molar equivalent dose of 2.5, 5, 10, and 20 mg/kg/wk were used for Pt-Mal-LHRH and carboplatin according to their molecular weights. After 2 weeks, end tumor volume was measured  and platinum tissue content in the tumor along with skeletal muscle was digested in aqua regia, diluted, and analyzed by inductively coupled plasma mass spectrometry . We used certified platinum standards for external standardization and picked all samples and standards with known concentrations of indium as an internal standard. All analysis included duplicates, matrix spikes and digestion blanks.Tissues were harvested from 12 wk old female Balb/c mice were snap frozen and stored in 10% TCA with acetone. Tissues were washed with acetone and homogenized in 2% SDS. After lysates were prepared and the proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membranes, which were then western blotted with the following antibodies: LHRH receptor , b-actin , respectively. The proteins were quantified using the ECL Clarity Max Western Blotting Substrate (Bio-Rad). The western blot images used for analysis are displayed in the supplemental material. Animal protocols were approved by the Committee of Animal Research Care and Use at Eastern Kentucky University.Intravenous injection of molar equivalent dose of 20mg/kg of carboplatin and Pt-Mal-LHRH was administered to the female Balb/c mice. After 2.5 hrs tissues were re-harvested for platinum analysis. A secondary study examining 24hr drug distribution was conducted with NCr nu/nu female mice by Charles River Laboratories . For both studies the platinum tissue content was analyzed as described above.6 cells. Molar equivalent dose of 10 mg/kg/wk were used for Pt-Mal-LHRH and carboplatin according to their molecular weights. Tumor growth was monitored for 2 weeks or with an endpoint tumor volume of 1500 mm³. All study data points are found in the supplemental material. In addition, all animals were euthanized under isoflurane anesthesia for terminal cardiac puncture blood collection and death assured via a cervical dislocation. The guidance set forth in the AVMA Panel on Euthanasia for all animals was followed. For anesthesia, the animals were anesthetized with isoflurane via induction box. They were maintained via nosecone isoflurane. Throughout the anesthetic period, they were monitored for lack of response to stimuli and appropriate cardiopulmonary function. Further, specific humane and study-derived endpoints were described in the IACUC protocols to ensure the welfare of the animal. If any animal presented with clinical issues or if unexpected outcomes were observed, the animal was referred to the Attending Veterinarian for diagnosis and treatment in consult with the study director. Sample sizes/cage of animals used were determined based off previous in-vivo experimental work with Pt-Mal-LHRH including the sample size needed for sufficient statistical relevance  and level of error during each experiment. Animal protocols and numbers were approved by the Committee of Animal Research Care and Use at Eastern Kentucky University (04–2018) and Charles River Laboratories .The MDA-MB-231 mouse tumor model was conducted at Charles River Laboratories for third party validation. Orthotopic tumor initiation was performed in female 12wk old NCr nu/nu mice through mammary pad injection of 5x10The data is illustrated as mean ± SEM and statistical analyses were performed using GraphPad, Prism 6 . Unpaired t-tests, along with, one and two-way ANOVAs were utilized as appropriate.To investigate the safety of therapeutic Pt-Mal-LHRH drug ranges, mice were administered by IV the doses 2.5, 5, 10, or 20 mg/kg. Pt-Mal-LHRH was found to significantly decrease the tumor growth, with 20mg/kg showing the largest amount of tumor regression . Tumor gTo determine the effectiveness of our Pt-Mal-LHRH compound we compared it to carboplatin since the drugs are structurally similar. The previous therapeutic dose ranges were used since no significant toxicological side effects were found. Pt-Mal-LHRH showed a significant attenuation in tumor growth compared to carboplatin and normal control growth , with the largest attenuation found with Pt-Mal-LHRH 20mg/kg treatment . Each PtTo examine the tumor targeting ability of LHRH the platinum concentration was examined in the tumor and skeletal muscle (control tissue). Pt-Mal-LHRH was found to have increased accumulation in the tumor tissue compared to skeletal muscle, indicating tumor targeting . MoreoveTo provide in-vivo tumor regression data, validation with a second TNBC cell type (MDA-MB-231) was investigated. Further, an external third party (Charles River Laboratories) was used to conduct the in-vivo tumor study. Since MDA-MB-231 are human derived, the NCr nu/nu immunocompromised mice were used and a dose of 10mg/kg was administered to ensure no toxicity was reached. Pt-Mal-LHRH was found to significantly decrease MDA-MB-231 tumor growth compared to carboplatin , supportThe design and synthesis of Pt-Mal-LHRH was developed to enhance the potency and accumulation of platinum within tumors overexpressing the LHRH receptor. Our prior work showed Pt-Mal-LHRH enhanced cytotoxicity at a cellular level due to receptor targeting, along with examining tumor regression through administration of an initial single dose . The curAdditionally, cancers afflicting reproductive areas including breast, prostate, ovarian, etc,, have been shown to overexpress the LHRH receptor, along with the TNBC cell lines 4T1 (mouse mammary carcinoma) and MDA-MB-231 (human mammary adenocarcinoma) , 26–29. Our Pt-Mal-LHRH compound design utilizes the LHRH hormone for the selective targeting of the LHRH receptor while reducing its systemic distribution and healthy cell targeting. Further, the cytotoxic effect of Pt-Mal-LHRH was previously shown to be induced by cellular entry of the Pt-Mal-LHRH compound binding to the LHRH receptor and a 20-fold increase in cellular uptake of Pt-Mal-LHRH was found compared to carboplatin due to overexpression of the LHRH receptor in 4T1 cells . Others Further, due to the tumor targeting ability of Pt-Mal-LHRH we found a significant decrease in tumor development in both our dose-range and carboplatin comparison studies. These experiments provided an important deeper examination of the therapeutic and safety profiles of Pt-Mal-LHRH. The investigated doses were chosen based on the therapeutic and toxicology profiles of carboplatin and cisplatin, since the formation of Pt-Mal-LHRH is a combination of two moieties, LHRH and activated cisplatin. Cisplatin is known to have a small therapeutic window with toxicity induced at 2.5–3 mg/kg and embryonic lethality at 5.24 mg/kg in mice, in contrast, carboplatin is known to be less toxic and can be used at higher doses up to 50 mg/kg –38. BothRegarding the tumor reduction efficacy of Pt-Mal-LHRH, there was a significant reduction in tumor development across the three independent mice studies conducted. In the therapeutic dose-range there was a significant decrease in 4T1 tumor volume across the 2.5–20mg/kg doses of Pt-Mal-LHRH . These dTogether, these data deliver further insights into the efficacy and targeting of Pt-Mal-LHRH as a treatment for TNBC by showing tumor reduction in two separate tumor models. The developmental purpose of Pt-Mal-LHRH was to provide a selective tumor targeting drug paradigm that would target tumors overexpressing the LHRH receptor while mitigating side effects commonly arising from chemotherapy. Our data indicate that drug conjugation using the LHRH hormone could be beneficial for drug delivery and modeling for chemotherapy treatment. Future studies are needed to further examine the toxicology and metabolism of the compound, including, the ability of Pt-Mal-LHRH to target other reproductive cancers overexpressing the LHRH receptor.Our results demonstrate that our synthesized Pt-Mal-LHRH chemotherapeutic compound can effectively target and attenuate TNBC tumor growth. This was validated by using two individual TNBC cell line tumor models. Further assessment of Pt-Mal-LHRH treatment showed no decrease in body weight or blood cell count below normal ranges due to toxicity. In addition, the distribution and tissue accumulation results showed targeting of areas overexpressing the LHRH receptor, thus, indicating selectivity towards these tissues while decreasing systemic accumulation, which could potentially mitigate side effects. Taken together, Pt-Mal-LHRH showed enhanced accumulation in reproductive tissues including the ovary, uterus, and mammary compared to carboplatin and effectively targeted TNBC tumors. The tumor targeting of Pt-Mal-LHRH led to selective tumor accumulation upon administration and enhanced attenuation of tumor growth compared to carboplatin. Subsequently, the results of our study support further investigation into the possible clinical use of Pt-Mal-LHRH for patients afflicted with triple negative breast cancer.While conducting in-vivo experimentation there can be issues that arise including injection error or reaching the designated tumor volume endpoint that can lead to study limitations and decreased statistical power. Injection errors were noted to take place in one animal in the 2.5mg/kg Pt-Mal-LHRH group in S1 File(DOCX)Click here for additional data file.S2 File(PDF)Click here for additional data file.


Although the extensive use of highly active oncological treatments has led to an increased rate of secondary resections in patients with colorectal liver metastases, it has led to questions regarding the correct surgical management of disappearing liver metastases (DLM) and small residual lesions during chemotherapy. Our study aimed to evaluate the clinical outcome of DLM and small remnant liver metastases (≤10 mm) assessed by means of hepatobiliary contrast-enhanced and diffusion-weighted MR imaging (DW-MRI) in metastatic colorectal cancer patients with first-line chemotherapy treatment in order to support the clinical management and decision-making process for these liver lesions. Our results showed that DLM assessed via hepatobiliary contrast-enhanced and DW-MRI very probably indicate a complete response in patients without chemotherapy-induced sinusoidal obstruction syndrome. In these patients, a follow-up with liver MRI can be considered, and resection should be performed in the case of disease relapse. The surgical removal of small remnants of liver metastases should always be advocated whenever technically possible.We aimed to evaluate the outcome of the disappearance or small remnants of colorectal liver metastases during first-line chemotherapy assessed by hepatobiliary contrast-enhanced and diffusion-weighted MR imaging (DW-MRI). Consecutive patients with at least one disappearing liver metastasis (DLM) or small residual liver metastases (≤10 mm) assessed by hepatobiliary contrast-enhanced and DW-MRI during first-line chemotherapy were included. Liver lesions were categorized into three groups: DLM; residual tiny liver metastases (RTLM) when ≤5 mm; small residual liver metastases (SRLM) when >5mm and ≤10 mm. The outcome of resected liver metastases was assessed in terms of pathological response, whereas lesions left in situ were evaluated in terms of local relapse or progression. Fifty-two outpatients with 265 liver lesions were radiologically reviewed; 185 metastases fulfilled the inclusion criteria: 40 DLM, 82 RTLM and 60 SRLM. We observed a pCR rate of 75% (3/4) in resected DLM and 33% (12/36) of local relapse for DLM left in situ. We observed a risk of relapse of 29% and 57% for RTLM and SRLM left in situ, respectively, and a pCR rate of about 40% overall for resected lesions. DLM assessed via hepatobiliary contrast-enhanced and DW-MRI very probably indicates a complete response. The surgical removal of small remnants of liver metastases should always be advocated whenever technically possible. Colorectal cancer (CCR) is the third-most-common cancer in both sexes and ranks second in terms of mortality worldwide . In mostDespite recent improvements in systemic treatment for metastatic colorectal cancer (mCRC) patients, resection of metastases remains the unique chance to cure or to prolong disease-free remission in selected patients with CRLM. The availability of active systemic therapies combined with growing advances in surgical techniques and the widespread multidisciplinary approach have broadened the group of patients deemed eligible for hepatic surgery ,12. WhilThe first imaging modality used to detect and characterize CRLM and evaluate resectability is usually contrast-enhanced computed tomography (CT). CT remains the workhorse and is the only useful tool in case of diffuse metastatic liver disease and/or metastatic extra-hepatic disease. In the last few years, liver magnetic resonance imaging (MRI) with hepato-specific contrast agents, such as gadoxetic acid and diffusion-weighted imaging, has been introduced for the management of patients with CRLM eligible for secondary resection . NotablyDrawing on these considerations, we evaluated the clinical evolution of DLM and small residual liver metastases (≤10 mm) assessed by means of hepatobiliary contrast-enhanced and diffusion-weighted MR imaging (DW-MRI) in metastatic CRC patients with first-line chemotherapy treatment in order to support the clinical management and decision-making process for these liver lesions.Consecutive patients with CRLM referred to Pisa University Hospital from November 2016 to December 2020 and with at least one DLM or residual liver metastases less than or equal to 10 mm, assessed via hepatobiliary contrast-enhanced and diffusion-weighted MR imaging during first-line chemotherapy, were included. Patients with disease progression were excluded.DLM in the case of vanished hepatic metastasis;Residual tiny liver metastases (RTLM) in the case of residual hepatic metastases ≤5 mm;Small residual liver metastases (SRLM) in the case of residual hepatic metastases >5 mm and ≤10 mm.Liver lesions included in the present study were categorized into three groups with reference to the baseline MR examination :DLM in tHepatic lesions larger than 10 mm were excluded from analysis.b values , parallel imaging technique and with diffusion-weighted gradients applied in all three orthogonal directions. Subsequently, a 3D breath-hold fat-suppressed T1-weighted LAVA (Liver Acquisition with Volume Acceleration) flex sequence was obtained before and after Gd-EOB-DTPA  injection, including both dynamic and hepatobiliary phases. Primovist was administered intravenously at a rate of 2 mL/s for a total dose of 0.1 mL/kg body weight, followed by a 25 mL saline washout.MR examinations were performed using a 3.0 T superconductive scanner . An 8-channel phased-array body coil was used for both excitation and signal reception. Our standard MR imaging liver protocol first included axial T2-weighted and in-/out-of-phase axial T1-weighted sequences. Axial DW-MRI was acquired through the entire liver using a single-shot spin-echo echo-planar (SE-EPI) sequence with multiple Lesion size was defined as the maximum diameter of the metastasis on the hepatobiliary phase imaging. Low signal intensity on T1-weighted image, moderate signal intensity on T2-weighted image, high signal intensity on DW-MRI and low signal intensity both on dynamic and hepatobiliary contrast-enhanced MRI were used as imaging criteria for detection of CRLM. In addition, sinusoidal obstruction syndrome (SOS), liver damage caused by treatment with oxaliplatin, was detected due to the presence of reticular hypointensity of liver parenchyma in the hepatobiliary phase. Imaging was evaluated by consensus reading of three radiologists , blinded with regard to clinical information and outcome.The outcome of resected liver metastases was assessed in terms of pathological response, while lesions left in situ were evaluated in terms of local relapse or progression according to RECIST criteria version 1.1. Tumor reassessments were performed every 8 weeks by means of CT scan; liver MRI was utilized as problem-solving diagnostic method.Descriptive statistics were used to summarize the pathological response of resected metastases. The cumulative relapse or progression rates at 6 and 12 months for non-resected DLM and residual metastases, respectively, were estimated using the Kaplan–Meier method, starting from the date of liver MRI performed during first-line chemotherapy until relapse or progression of each lesion. Liver lesions not relapsed or progressed at last follow-up visit or at the beginning of a subsequent line of chemotherapy  were censored at the date of the last radiological assessment. Log-rank test was used to compare the cumulative relapse rate based on the presence of SOS in DLM group. A two-sided p-value less than 0.05 was considered statistically significant. All statistical analyses were carried out using MedCalc version 14.8.1 .Among 120 patients with CRLM undergoing liver MRI, 64 patients performed this exam during first-line chemotherapy. Fifty-two patients had at least a DLM or a residual metastasis less than or equal to 10 mm and were included in our analysis .Clinical characteristics of the patient population are summarized in First-line chemotherapy regimens used were mostly triplet plus anti-VEGF or anti-EGFR in 16 patients (31%) and doublets plus anti-EGFR in 15 patients (29%), while 13 (25%) and 8 (15%) patients were treated with doublets plus anti-VEGF and doublets alone, respectively.Out of 265 liver lesions radiologically reviewed, 185 metastases fulfilled the inclusion criteria of this study: 40 DLM, 82 RTLM and 60 SRLM, as reported in Overall, 15 patients had at least a DLM. The median number of DLM was 1.5 (IQR: 1–3). The median time interval between diagnosis of liver lesions at baseline MRI and the detection of DLM at MRI was 6.6 months (IQR: 4–8.7 months). Among 40 DLMs, the site where DLM were located was resected in four cases (two in one patient and another two in two different patients). A complete pathological response was observed in three (75%) instances .p = 0.003) , with 6- and 12-month cumulative relapse rates of 26% and 43%, respectively , panel A= 0.003) , panel BOverall, 33 patients had at least an RTLM . The medOverall, 29 patients had at least an SRLM . The medFor decades, CRLM were listed as a non-curable medical finding. Surgical techniques for liver surgery, systemic treatment regimens and perioperative care have significantly improved over time. Systemic chemotherapy and targeted therapy have significantly increased the chance of conversion to surgery for patients with initially unresectable colorectal liver metastases, resulting in improved survival. The management of DLM and small remnant of liver metastases after chemotherapy has always been a matter of debate ,20. ReceSeveral retrospective studies addressed this question . AlthougRegarding the small remnants of liver metastases after chemotherapy, our results showed a risk of relapse of 29% and 57% for RTLM and SRLM left in situ, respectively, and a pCR rate of about 40% overall for resected lesions . SimilarIn our series, all MR exams were performed with a state-of-the-art magnet operating at 3.0 T, and a multiparametric MR imaging protocol, including both diffusion-weighted imaging and hepatobiliary phase, was utilized. In this way, very small liver metastases could also be identified. However, MRI is very susceptible to breathing artefacts, especially in the segments of the hepatic dome; in the case of significant artifacts, the evaluation of liver parenchyma may be not reliable, and the detection of small tumor foci can be extremely difficult. As suggested by Beyer et al. , in thesOur study has several limitations, including the retrospective nature, the low sample size, the limited follow-up and the use of CT scan as follow-up imaging for non-resected lesions that could have underestimated the risk of relapse.Although no definitive conclusions can be drawn, our results, in addition to the literature data, can provide same suggestions for the proper management of DLM and small remnants of liver metastases after chemotherapy. In particular, since DLM assessed through hepatobiliary contrast-enhanced and diffusion-weighted MRI indicates a real complete response with high probability in patients without chemotherapy-induced SOS, follow-up without resection can be considered for these cases ,35. IndeThe treatment plan for patients with CRLM should be determined case by case in a multidisciplinary setting. Adequate radiological staging and restaging after chemotherapy are crucial for the optimal selection of patients as candidates for surgery and to individualize the optimal treatment strategy. DLM assessed via hepatobiliary contrast-enhanced and diffusion-weighted MRI very probably indicates a complete response in patients without chemotherapy-induced SOS. In these patients, a follow-up with liver MRI can be considered, and resection should be performed in the case of disease relapse. The surgical removal of small remnants of liver metastases  should always be advocated whenever technically possible.


The apolipoprotein B mRNA-editing catalytic polypeptide-like (APOBEC) family specifies multiple cytidine deaminases. In mammals, a minimum of 5 genes encode APOBECs 1, 2, 3, 4, and activation-induced cytidine deaminase (AID) . This exAlthough deamination of retroviral DNA is likely the primary mechanism of viral inhibition, deamination-independent modes of APOBEC activity have been observed ,13. BecaThe betaretrovirus mouse mammary tumor virus (MMTV) has provided a model for the first in vivo studies of APOBEC3 activity . MMTV reC  motifs in MMTV proviruses that are unable to produce Rem are heavily mutated in BALB/c mice to produce stop codons compared to proviruses that express Rem [-/-Aicda mice with Rem-mutant MMTV. Infection of Aicda-/- mice greatly reduced proviral mutations in WRC motifs, which are known AID-targeting sequences in nuclear immunoglobulin genes [C-target motifs  in MMTV proviruses observed in BALB/c mice also increased in the absence of Rem but, surprisingly, the mA3-type mutations in Rem-null proviruses disappeared when AID was absent. Furthermore, coexpression of Rem in tissue culture cells with either AID or mA3 resulted in proteasomal degradation of AID, but not mA3 [WRress Rem . This diin genes . In contin genes . The mA3 not mA3 . These r not mA3 . MMTV Re not mA3 , consistIn contrast to other APOBEC enzymes, the roles of A2 and A4 proteins remain mysterious. Recent data suggest that these deaminases may have evolved from enzymatic activities to sole use of their nucleic acid-binding properties. Recent work has hinted that A2 and A4 bind to promoter regions to affect transcriptional activity of selected genes . AlternaThe answer most certainly is affirmative. Ample evidence indicates that replication of viruses in APOBEC-expressing cells has led to the appearance of novel viral variants . As noteIn addition to the known effects of APOBECs on HIV-1 variation acting at the level of DNA replication , the CorAlthough SARS-CoV-2 and Ebola are positive and negative sense RNA-containing viruses, respectively, both have been implicated as cross-species transmission events to humans, perhaps from bats . Bats arThe regulation of APOBEC expression is only partly understood. APOBEC cytidine deaminases are part of the innate immune system, yet multiple reports indicate that other functions of innate immunity, such as signaling through the type I IFNs and Toll-like receptors (TLRs), increase levels of the A3 enzymes –39. The Regulation of AID expression reveals the multiple pathways that control APOBEC transcript levels. Treatment of cultured B cells with lipopolysaccharides (LPS)  in conjunction with the cytokine interleukin-4 (IL-4) is known to induce AID . In B ceEvolutionary evidence suggests that APOBECs are advantageous because of the expansion of this gene family in higher organisms and the amplification of the A3 genes in primates. Many of these genes appear to be under positive selection ,30. The Matonaviridae) and SARS-CoV-2 (Coronaviridae), have C-to-U editing typical of APOBECs [C motifs typical of APOBECs. This study also indicated that the vaccine-derived rubella quasi-species were recovered from the nasal cavity, but not in the bloodstream, suggesting that APOBEC-induced mutations could serve as a source of novel viruses for respiratory transmission [Although previous reports have suggested that viruses with DNA replication intermediates are the most affected, more recent experiments argue that RNA-containing viruses, such as rubella  scenario emerges for the role of APOBECs in human cancers, of which approximately 10% are due to viral infections . Interesmyc [Current databases have designated nuclear APOBECs, such as A3A, A3B, and AID, as relevant to human cancer . A3A hasmyc . In addimyc , perhapsmyc . Recentlmyc . These dIn summary, APOBECs induce mutations in both viral and host DNA, but it appears increasingly likely that RNA-containing viruses are mutated by cytidine deaminases. These mutations limit viral replication and transmission. APOBEC-mediated changes on host mRNAs in innate immune cells may provide additional short-term adaptations to inhibit viral infections. Nevertheless, these mechanisms yield viral selection. APOBEC expression in cancers leads to DNA damage and tumor cell death, yet allows for immune escape variants. Indeed, APOBECs are fickle friends.


C. elegans genomics-genetics screens identifies trafficking molecules that are not implicated in apical sorting yet polarize apical membrane and PAR complex components. Live tracking of polarized membrane biogenesis indicates that the biosynthetic-secretory pathway, linked to recycling routes, is asymmetrically oriented toward the apical domain during this domain’s biosynthesis, and that this directionality is regulated upstream of PARs and independent of polarized target membrane domains. This alternative mode of membrane polarization could offer solutions to open questions in current models of epithelial polarity and polarized trafficking.In prevailing epithelial polarity models, membrane-based polarity cues  position apicobasal cellular membrane domains. Intracellular vesicular trafficking expands these domains by sorting polarized cargo toward them. How the polarity cues themselves are polarized in epithelia and how sorting confers long-range apicobasal directionality to vesicles is still unclear. Here, a systems-based approach using two-tiered  Biosynthetic trafficking polarizes epithelial membranes by asymmetrically inserting the apical domain into the growing membrane. Current epithelial polarity models order the highly conserved extracellular-, intracellular-, and membrane-based polarity cues sequentially during the process of cellular polarization: (i) Extracellular matrix signals orient cells within the epithelium; (ii) plasma membrane– and junction-based apical and basolateral core polarity determinants  specify apicobasal domain positions at the plasma membrane; (iii) intracellular vesicular trafficking expands and maintains these domains by supplying polarized membrane components (Caenorhabditis elegans (C. elegans) intestine. Reducing and restoring GSL biosynthesis reversibly shifts apical membrane components, junctions, PARs, and the lumen between apical and basolateral sides of growing cells that no longer divide and move but still expand their plasma membranes  (We previously demonstrated that glycosphingolipids (GSLs), saturated obligate membrane lipids, function in apicobasal membrane polarity in the C. elegans intestine  and unicellular  C. elegans tubular organ epithelia , through late embryogenesis and four larval stages , to the adult   has been complicated by the flatness of epithelial cells and by tissue complexity, as well as by confounding effects of polarized cell migration and division that coincide with de novo polarized membrane biogenesis. To overcome these limitations, we here examined de novo polarized membrane biogenesis live in single postmitotic cells at their invariant positions within the three-dimensional (3D)  context of single-layered, expanding pithelia . We trac+ membranes along apicobasal cellular axes between cells during inTERcellular lumenogenesis in the intestine and along anterior-posterior cellular axes during inTRAcellular lumenogenesis inside the single canal cell. This approach permits the distinction of the development of apicobasal membrane polarity from other aspects of apical membrane biogenesis by discerning (i) apical membrane assembly , (ii) apical membrane positioning , and (iii) apical membrane expansion .We tracked ERM-1::GFPC. elegans screens  from other tubulogenesis phenotypes and sorted them into 10 classes by their effects on (i) apical membrane assembly . As expected, bona fide apicobasal membrane polarity defects, here narrowly defined as defects in apical membrane positioning .In several RNA interference (RNAi)–based visual C. elegans trafficking genes  suggested that polarity conversion was suppressed by reducing the basolateral misrouting of apical membrane components  and 2 ; reversion proceeds in the reverse order; fig. S6A]. Monitoring membrane expansion in double C. elegans intestine, in a let-767(−/−) background (fig. S6C). Depletion of most of these molecules failed to suppress the let-767–dependent polarity conversion but identified two more suppressors (the AP-2 adaptor DPY-23 and the ARF-like GTPase ARL-8), implicating the disruption of specific endocytic routes, but not the general disruption of endocytosis or vesicle degradation, in the mechanism of suppression. We also failed to find evidence for a suppressor-dependent loss of lysosomal GSL degradation that might have been expected to suppress polarity conversion by increasing GSL levels . If, in contrast, suppression was caused by the loss of gene-specific trafficking functions, increasing these functions should enhance polarity conversion. Consistent with this prediction, expression of the constitutively active RAB-7Q68L (let-767(RNAi) intestines  anterograde basolateral trafficking routes downstream of GSL-directed apical sorting/routing sites  rather than apical membrane positioning  expanding postmitotic cells at their fixed positions in growing larval intestines. ERM-1 and ACT-5/actin were found to be increasingly retained on ERM-1+ and ACT-5+ cytoplasmic vacuolar inclusions but not misdirected to basolateral domains during de novo apical membrane biogenesis  larval intestinal cells, we used serial transmission electron microscopy (TEM) sectioning . This analysis failed to detect vacuoles with inward-pointing microvilli, suggesting that the observed vacuolar inclusions were endomembrane intermediates en route to or from the apical membrane and not ectopic lumens indicative of apicobasal polarity conversion. These results were consistent with a function of GSL suppressors in wild-type apical membrane assembly but not positioning.To confirm that the three GSL suppressors affected apical membrane assembly  domain in wild-type larval intestinal cells  secretory routes, moving unsorted apicobasal cargo toward GSL-dependent sorting/routing sites, as part of, or parallel to, the GSL-directed trajectory  retained the recycling marker hTAC::GFP on cytoplasmic vesicles in larval intestinal cells  larval intestinal cells, GFP::RAB-11+ vesicles were largely retained at expanding apical membranes, while GFP::RAB-10+ vesicles aggregated in the cytoplasm and were lost from basolateral membranes  and RME-1+ (presumed membrane-directed) endocytic-recycling carriers . DAB-1, RAB-7, and VHA-6, although themselves dispensable for apical domain positioning, support GSLs in this polarity function via their canonical roles in endocytic recycling that here serve to supply the GSL-directed secretory route that positions the nascent apical domain. Reducing influx into the GSL-directed apical vesicle trajectory reduces basolateral misrouting of apical membrane components upon GSL depletion and hence suppresses the GSL-dependent apicobasal polarity conversion  directionality of bulk membrane delivery to polarize the epithelial membrane  and junctions must themselves be polarized to membrane domains whose positions they ought to determine. The well-characterized signaling network of mutual inhibition between these canonical, apical, and basolateral membrane-based polarity cues . The proA flexible directionality of the secretory pathway, regulated independent of cargo sorting, also addresses difficulties of sorting-based models of polarized trafficking that place the directional control of trafficking with polarized cargo signals and their carrier-based decoders  the apical domain and associates with polarized RAB-11+ vesicles before PARs are polarized (C. elegans intestine (Drosophila follicular epithelium)  proceeds similarly between pairs of cells , it will position the apical, and consequently the basolateral, domain (fig. S9). Consistent with this idea, all trafficking components that we found to regulate polarity in the intestine also direct apical membrane expansion in the canal . In support of this observation, we note that several trafficking molecules have been identified by a function in the polarity of multicellular tubular epithelia that were independently identified by a function in the expansion of unicellular tubular epithelia  . Notably, inTRAcellular lumenogenesis models propose the expansion and polarization of the apical/lumenal membrane by directional vesicle coalescence It has been noted that secretory pathway components are conspicuously absent from the collection of trafficking molecules identified in numerous genetic screens on apicobasal polarity  (C. elegans) and in vitro (MDCK cells) models may thus provide complementary vistas on the same trafficking-directed process of membrane polarization, with apical recycling molecules such as RAB-11/Rab11  marking shared components of one common apical secretory trajectory that traverses the recycling compartment. A developmentally regulated, directionally flexible biosynthetic-secretory pathway, in close apposition to polarized recycling routes that permit exchange between all membrane domains, would provide greater plasticity of single-cell polarity than currently appreciated.While anterograde secretory routes can intersect with recycling routes on their way to the membrane, retrograde recycling routes can intersect with pre- and post-Golgi secretory routes, an intersection thought to, respectively, replenish pre-Golgi intermediates and increase the fidelity of cargo sorting  OP50 (Wild-type (N2 Bristol) and mutant E. coli HT115 (DE3), producing double-stranded RNA (dsRNA) of the gene of interest, as previously described  and carbenicillin (25 μg/ml). dsRNA was induced at room temperature for at least 6 hours before picking four to six L4 larvae onto each RNAi plate. Most bacterial clones were derived from the Ahringer genome-wide RNAi feeding library . The integrity of all RNAi clones was verified by sequencing (discrepancies to reported sequence information are noted in table S1).RNAi was carried out by feeding worms rrf-3(pk1426) (gfp RNAi on a green fluorescent protein (GFP)–expressing strain].RNAi was the method of choice for the here performed systems-based loss-of-function analysis of essential genes : It (i) produces a broad range of phenotypes, (ii) can be titrated to induce very mild to severe effects, and (iii) effectively targets maternal RNA . In tier-1 screens, moderately severe (standard) RNAi conditions were used to generate a balanced spectrum of phenotypes. To avoid full disruption of membrane-directed trafficking, a scaled-intensity RNAi approach was used to analyze the secretory pathway  cystic short canal phenotype (N = 1846). Modification of the ERM-1[++] excretory canal defect  was assessed under a dissecting fluorescent microscope in live animals carrying an integrated transgene, providing additional erm-1 copies (fgIs2[perm-1::erm-1]) and an extrachromosomal transgene labeling the canal cytoplasm .Screens I and II, genome-wide RNAi-based intestinal and excretory canal tubulogenesis screens  ,henotype  (28), exC. elegans or other species, based on published experimental data and search engines . Fifty trafficking genes were identified. All 50 gene products were highly conserved from yeast to human. Gene products were classified according to structure and trafficking function and annotated with regard to their yeast and human orthologs, cellular and subcellular localization, and, where available, trafficking route, with specific attention to functions described in C. elegans (collected in table S1).Subsequent selection steps used in this study (fig. S2) were carried out as follows: (i) From all tubulogenesis phenotypes identified in screens I to III, those phenotypes were selected that had defects in “apical/lumenal membrane assembly, positioning or expansion” ; (ii) after unblinding the identities of all genes whose losses caused such defects in apical membrane biogenesis, those genes were selected that had been implicated in vesicular trafficking. All genes were selected whose direct or homologous gene products had reported functions in vesicular trafficking in n > 5, with N > 500 animals per plate), performed in triplicates. Animals were characterized alongside a positive control  and a negative control . Note that all negative results of the initial tier-1 screens   larvae, was evaluated. The dominant marker rol-6, conferring a circumferential rolling movement, facilitates the 3D  evaluation of polarized tubulogenesis. RNAi conditions were empirically determined and varied to (i) track polarized membrane biogenesis throughout the entire postmitotic growth phase (see fig. S1) and (ii) examine a spectrum of mild-to-severe loss-of-function phenotypes. All animals were evaluated throughout development by high-power dissecting and/or confocal microscopy, with most phenotypes present in embryos and/or early larvae. For the initial assessment of lethality (table S1), the F1 progeny was counted on day 3. The morphology of embryos was assessed by differential interference contrast (DIC) (Nomarski) microscopy . Sterility was scored based on the number of total F1 progeny per plate, and embryonic morphology defects/larval arrests were counted per 100 embryos/larvae, respectively.For the standard analysis of RNAi phenotypes, the progeny of two RNAi plates, seeded with four L4 stage In the repeat RNAi analysis of the 50 identified trafficking genes, all RNAi clones were examined in two rounds: one focusing on embryonic, the other on early larval development. Intestinal and canal apical/lumenal membrane biogenesis was assessed in parallel in the same animal. Absence/presence of specific phenotype subclasses  was examined in separate sets of experiments using empirically determined gene-specific RNAi conditions, as described above.For the extended RNAi analysis of apical membrane assembly, positioning, and expansion defects, embryonic and larval intestinal and excretory canal tubulogenesis and ERM-1::GFP placement were evaluated 4 to 6 hours over day during the window of ~48 hours required for full tube extension , throughout the process of net polarized membrane expansion (see fig. S1). Phenotypes that occurred later (≥72 hours) were not included, given the high percentage of nonspecific late effects caused by membrane biogenesis defects. For example, intestinal membrane polarity defects  can be obscured during polarized membrane expansion by increasing cytoplasmic ERM-1::GFP displacement, the development of structural apical/lumenal membrane defects , vacuolar ERM-1::GFP displacement, and other tubulogenesis defects.let-767(s2819) mutants balanced with the free duplication sDp3 and labeled with the ERM-1::GFP transgene were used for the genetic interaction screens . Ectopic lumens in let-767(RNAi) intestines were scored by staining for apical intermediate filaments using the anti–IFB-2 antibody MH33  or rrf-3(pk1426)II; fgEx13 transgenic animals. More than 20 RNAi plates (60 × 15 mm) were set up with five L4 larvae, and progeny was allowed to develop to the mid-larval stage if not arrested earlier. Before harvesting the progeny, animals were checked under a fluorescent dissecting scope for the presence of the phenotype . Larvae were washed off in standard M9 medium . Results reported here reflect one set of quantification experiments performed with L3-stage larvae . For fluorescence intensity measurements, a square area of 250 × 200 pixels was chosen in the anterior intestine, corresponding to four cells (INT II and INT III), and ImageJ [National Institutes of Health (NIH)] software was used for image analysis. For quantification of vesicles, two circles of 100 pixels of the same area were counted.All images were analyzed by ImageJ software. To determine membrane versus cytoplasmic fluorescence intensity ratios between different animals, identical confocal laser settings were used for each set of experiments. Fifty-pixel long lines were drawn orthogonally across each of the four anterior-most lateral membranes between the first-to-second and second-to-third intestinal rings (INT II and III). The mid-point of the line was always placed on the lateral membrane. The medians of the maximum fluorescence intensity values were used for both membranes and the cytoplasm, and the membrane/cytoplasm ratios of the four measured cells were averaged and compared to controls. The average of four membrane/cytoplasm ratios was calculated, and more than seven sets of serial confocal images were analyzed. The average from three independent experiments was used to draw the bar graph. For quantification of the PEPT-1::DsRed, PAR-6::GFP, GFP::PKC-3, and GFP::RAB-10 membrane/membrane-associated/cytoplasm fluorescence intensity ratio, three cells of each worm were used for fluorescence intensity quantification and >10 worms were used to calculate the final membrane/cytoplasm intensity ratio. For quantification of the presence/absence of hTAC on the plasma membrane, the four lateral membranes between the first three anterior intestinal INT rings (INT I/II and INT II/III) of suppressor knockdowns versus controls were examined for the absence, reduction, and presence of hTAC. More than 30 animals were counted per experiment, and each experiment was repeated three times. Note that quantification of fluorophore loss on basolateral membranes  underestimates the actual loss since only a fraction of membranes retain sufficient fluorophore to qualify for quantification.Animals labeled with fluorescent endomembrane and plasma membrane components were scanned by confocal microscopy . Per worm, fluorescent puncta were counted separately inside two intestinal cells, outlined by a fluorescently labeled basolateral membrane marker. Puncta were subjectively determined as endosomal vesicles, considering factors such as shape, size, boundary, and signal intensity . Vesicles were classified into three categories: membrane-associated (puncta overlapping with plasma membrane components), tubular (puncta in connection to a cytoplasmic tubular structure that may or may not be connected to the membrane), and cytoplasmic (puncta separated from surrounding structures). Eight animals were counted per each experiment, and more than three independent experiments each were performed.N (sample size) and n (number of independent experiments) are indicated in the text and figure legends. *P < 0.05, **P < 0.01, ***P < 0.001.Statistical analyses were performed by GraphPad Prism 5 software, and the specific tests used for each experiment are listed in the figure legends. All values shown are mean ± SEM of three or more independent experimental data sets. Where pertinent,