An increase in cardiac index (CI) during an end-expiratory occlusion test (EEOt) predicts fluid responsiveness in ventilated patients. However, if CI monitoring is unavailable or the echocardiographic window is difficult, using the carotid Doppler (CD) could be a feasible alternative to track CI changes. This study investigates whether changes in CD peak velocity (CDPV) and corrected flow time (cFT) during an EEOt were correlated with CI changes and if CDPV and cFT changes predicted fluid responsiveness in patients with septic shock.Prospective, single-center study in adults with hemodynamic instability. The CDPV and cFT on carotid artery Doppler and hemodynamic variables from the pulse contour analysis EV1000™ were recorded at baseline, during a 20-s EEOt, and after fluid challenge (500 mL). We defined responders as those who increased CI ≥ 15% after a fluid challenge.r = 0.51 [0.26–0.71]). A significant, albeit lower correlation, was found for cFT (r = 0.35 [0.1–0.58]). An increase in CI ≥ 5.35% during EEOt predicted fluid responsiveness with 78.9% sensitivity and 91.7% specificity, with an area under the ROC curve (AUROC) of 0.85. An increase in CDPV ≥ 10.5% during an EEOt predicted fluid responsiveness with 96.2% specificity and 53.0% sensitivity with an AUROC of 0.74. Sixty-one percent of CDPV measurements (from − 13.5 to 9.5 cm/s) fell within the gray zone. The cFT changes during EEOt did not accurately predict fluid responsiveness.We performed 44 measurements in 18 mechanically ventilated patients with septic shock and without arrhythmias. The fluid responsiveness rate was 43.2%. The changes in CDPV were significantly correlated with changes in CI during EEOt .The online version contains supplementary material available at 10.1186/s13054-023-04422-9. Fluid responsiveness is defined as an increase in cardiac index (CI) in response to an increase in preload . In patiSeveral functional hemodynamic tests for assessing fluid responsiveness have been introduced in clinical practice in the last few years . These tThe EEOt is based on an interruption of mechanical ventilation at the end of expiration for 15–30 s. This maneuver increases cardiac preload, venous return, and stroke volume (SV) in preload-responsive patients. In one study , an incrWe conducted this study to assess (1) if there was a correlation between the changes in carotid Doppler peak velocity (CDPV) or in corrected flow time (cFT) and the changes in CI induced by a 20-s EEOt; (2) if EEOt-induced changes in CDPV and cFT predict fluid responsiveness in patients admitted to intensive care unit (ICU) with septic shock.The institutional review board of Policlinico Agostino Gemelli University Hospital approved the present study (Prot. N. 0048329/18 ID 1677) retrospectively registered on ClinicalTrials.gov NCT04470856 on July 14, 2020. All patients or their legal representatives gave their written informed consent to participate.All adult patients (≥ 18 y) admitted to the general ICU of Policlinico Agostino Gemelli University Hospital with a diagnosis of septic shock who were sedated and mechanically ventilated were considered for inclusion. Patients were eligible if they needed a fluid challenge according to predefined clinical criteria: (1) hypotension  and/or (2) tachycardia  and/or (3) urinary flow ≤ 0.5 mL/kg/min for 2 h. Those with significant valvular heart disease, cardiac arrhythmia, heart failure, peripheral arterial disease, or common carotid artery stenosis narrower than 50% were excluded from the study.All patients were supine with their trunk elevated 30°, sedated, paralyzed, and mechanically ventilated in the volume control mode . We ascertained the absence of spontaneous breathing activity by evaluating the flow/pressure curves displayed on the ventilator screen. Tidal volume was set at 6 mL/kg of predicted body weight (PBW).We measured CI using transpulmonary thermodilution (TPTD) . CI and the other hemodynamic variables derived from pulse contour analysis were recorded over 20 secs . Before EEOt consisted of a prolonged expiratory pause (20 s) in which no expiratory or inspiratory efforts were recorded.Fluid responsiveness was defined as an increase in CI of more than 15% after a standardized fluid challenge (500 mL of Ringer’s lactate in 10 min).Fluid responders were tested more than once, at least 12 h apart, if they met the inclusion criteria again. Each EEOt was considered a single and independent measure.The following protocol describes our procedure in detail Fig. .Fig. 1StT1 (baseline 1): we performed the first set of TPTD to assess the CI, the SVI, the stroke volume variation (SVV), and the systemic vascular resistance index (SVRI). We also recorded heart rate (HR), mean arterial pressure (MAP), and central venous pressure (CVP). Simultaneously, we measured the CDPV and the cFT using carotid Doppler (see below).T2 (EEOt): one operator applied a 20-s EEOt and measured the hemodynamic variables using the pulse contour method, while a second operator performed a carotid Doppler and recorded CDPV and cFT during the last five cardiac cycles before the end of EEOt.T3 (baseline 2): we visually verified that the hemodynamic values had returned to the baseline before administering the fluid challenge.T4 : the patients received a 500-mL bolus of Ringer’s lactate solution in 10 min. After completing the fluid challenge, we performed the last series of hemodynamic measurements, including CI, MAP, HR, SVI, CVP, SVRI, and carotid Doppler, as above.Two operators collected the hemodynamic variables manually and kept a screenshot of the measurements as a record for data check. Catecholamine infusion, mechanical ventilation settings, and bed position were kept constant at all times.One investigator (SD'A) with previous formal training in critical care ultrasound insonated the common carotid artery in a longitudinal view approximately 1–2 cm before the bifurcation using a 5–10-MHz linear array transducer  and performed a pulse-wave Doppler analysis. The sample volume was in the vessel’s middle, with the caliper positioned parallel to flow, angled at no more than 60°. We averaged five consecutive measurements to limit the effects of respiratory-induced changes in CDPV and cFT.pre values refer to the Baseline 1 and the CPDVpost values refer to those obtained during EEOt or after fluid challenge.The ΔCDPV was expressed as a percentage and was calculated with the following formula:The FT was measured from the beginning of the flow tracing’s upstroke to the nadir of its dicrotic notch on a pulse waveform analysis. We used the Wodey’s formula: cFT = FT + 1.29*(HR-60) to correct measurements for the heart rate . We measThe primary endpoint of this study was the correlation between changes in CDPV and CI during an EEOt. Secondary endpoints were the correlations between changes in CDPV and CI after the fluid challenge and between changes in cFT and CI during EEOt and after fluid challenge. We also explored the ability of changes in CDPV and cFT during EEOt to predict fluid responsiveness. Differences between responders and non-responders were studied during all three phases of our observation .The distribution of continuous variables was assessed with the Kolmogorov–Smirnov test. Data are expressed as mean ± standard deviation (SD) or median (interquartile range), as appropriate. Categorical variables are reported as fractions and percentages (%). Differences between parametric variables were evaluated with the Student’s t test for unpaired and paired measures or with the Mann–Whitney and Wilcoxon tests, as appropriate. Fisher’s exact test was adopted for categorical variables. LSD and Scheffé’s test were used to correct for multiple comparisons.J = sensitivity + specificity − 1).We used two-way ANOVA to detect significant variable changes across time points between two groups. We calculated a receiving-operator characteristic (ROC) curve using the Wilson–Brown method to summarize the ability of each measure to predict fluid responsiveness across the various thresholds. For all predictors, we identified the optimal threshold based on the Youden index J , we considered an AUROC of 0.75, a null hypothesis of 0.5, and beta and alpha error as above. The total required sample was 40 measurements.For the carotid Doppler variables, we calculated how many measurements fell within the gray zone, i.e., the number of cases with a sensitivity < 90% and/or a specificity < 90%.We also estimated the precision and the least significant change (LSC)  of CDPV.Precision was estimated asi et al. The LSC was calculated as(rmcorr)  between (rmcorr) . In theshttps://www.medcalc.org; 2022), and the rmcorr R package (https://cran.r-project.org/web/packages/rmcorr/) for statistical analysis.We used SPSS® 28 software (IBM Chicago IL), MedCalc® Statistical Software version 20.210 .2; p = 0.012) and a higher SVV  than non-responders. There were no significant differences in HR, MAP, CVP, and SVRI. Fluid responders also had a significantly lower CDPV  and cFT  than non-responders  and the CDPV increased by 10.5%  in responders, while no significant change was observed in non-responders  and the CDPV increased by 19.6% , while no significant change was observed in non-responders  , while no significant correlation was found in non-responders  was greater for cFT than for CDPV at EEOt   Fig. A.Fig. 4Ap = 0.006)  and 53.0% sensitivity (95% CI 32.1–73.3), with an AUROC of 0.743  or SV (ρ = 0.95). However, the results of that study are not directly applicable to the critical care setting. Moreover, that study used a different carotid Doppler equipment (wearable device) and evaluated a different index (changes in carotid VTI pattern) compared to our study.To the best of our knowledge, this is the first study assessing fluid responsiveness using the carotid Doppler variations in association with an EEOt in critically ill patients. The potential usefulness of the changes in carotid Doppler during EEOt has been previously investigated in healthy volunteers . The autTwo studies , 19 inveEEOt and fluid challenge were not consistently associated with increased Doppler velocity. Namely, CDPV decreased in a minority of responders  and almost half of the non-responders . Regarding PEEP, one study showed that PEEP levels ranging from 5 to 14 cmH2O do not affect the predictive ability of the EEOt [Results of the repeated measures correlation clearly showed that the changes in CDPV and cFT both during EEOt and after fluid challenge were correlated with the parallel changes in CI or SVI in responders. This is consistent with the ability of EEOt-induced changes in CPDV to predict fluid responsiveness in our study. However, the accuracy of CDPV was lower than that of other indices, such as PLR. In fact, the gray zone of CPDV was 61%, meaning that only the CDPV values included in the remaining 39% range were sufficiently accurate to make a clinical decision. A possible explanation for this finding may be that the EEOt causes smaller changes in venous return compared to the PLR. The PLR in fact causes a rapid displacement of a high volume about 300 mL) of venous blood from the lower body toward the right heart, mimicking an entire fluid challenge . Another0 mL of vThe clinical concordance analysis of the percentage changes in Doppler variables vs. the percentage changes in CI showed that many of the concordant changes in cFT were ± 5% or less. Compared to cFT, CDPV had a greater proportion of clinically significant concordant changes . Carotid Doppler could therefore be used to investigate the relationship between cardiac contractility and vascular resistance in response to therapy, assuming that CDPV measurement is accurate and that blood flow in the supra-aortic vessels mirrors the blood flow in the thoracic aorta.Fluid responsiveness depends on the slope of the Frank–Starling curve, which is critically affected by cardiac contractility. In our study, we excluded patients with cardiac failure, so that the role of cardiac contractility was not specifically assessed. However, previous observations made using aortic Doppler  have shoSome limitations of our study must be acknowledged. Firstly, this study was conducted on adult patients with septic shock in controlled ventilation with 6 mL/kg of PBW with absent or minimal spontaneous breathing, and its findings are not necessarily generalizable to other patient populations. Secondly, this technique is based on manual detection and—as such—is prone to intra-observer variation. In our study, all carotid Doppler measurements were made by a single experienced operator. While this was essential to ensure the reproducibility of our measurements, it may have overestimated their precision and reduced the external validity of our findings. Thirdly, we used the EV1000™/Volume View device, which calculates SVI and CI on the average pulse contour recorded over 20 s. Inevitably, this time window only partially overlapped with those of the EEOt, which may have affected the accuracy of our findings. Fourthly, as specified in our Methods, we compared both the hemodynamic measurements made during EEOt and those made after the fluid challenge with the first baseline (T1), since on T3 the return to the baseline after the EEOt was assessed only visually. Although the effect of the EEOt is assumed to be transient, we cannot exclude that some residual effects may have persisted after the end of EEOt and have reduced the accuracy of the subsequent measurements. Fifthly, the 61% gray zone we found in our study suggests caution when using Doppler results to make clinical decisions. Lastly, carotid Doppler might be less accurate in patients affected by arrhythmias, such as atrial fibrillation, which are common in septic shock patients , or by vOur study showed that in adult patients with septic shock, the changes in CPDV and cFT during EEOt on carotid Doppler are correlated with the changes in CI that occur after a fluid challenge, and that an increase in CDPV greater than 10.5% during a 20-s EEOt predicted fluid responsiveness with > 95% specificity. Carotid Doppler can be used to assess fluid responsiveness in patients who are sedated and mechanically ventilated and without arrhythmias, when invasive hemodynamic monitoring is not immediately available. However, its large gray zone is an important limitation.Additional file 1. Supplementary figures.


Approximately 50% of patients with primary colorectal carcinoma develop liver metastases. This study investigates the possible molecular discrepancies between primary colorectal cancer (pCRC) and their respective metastases.A total of 22 pairs of pCRC and metastases were tested. Mutation profiling of 26 cancer-associated genes was undertaken in 22/22primary-metastasis tumour pairs using next-generation sequencing, whilst the expression of a panel of six microRNAs (miRNAs) was investigated using qPCRin 21/22 pairs and 22 protein biomarkers was tested using Reverse Phase Protein Array (RPPA)in 20/22 patients’ tumour pairs.TP53 (86%), APC (44%), KRAS (36%), PIK3CA (9%), SMAD4 (9%), NRAS (9%) and 4% for FBXW7, BRAF, GNAS and CDH1. The primary-metastasis tumour mutation status was identical in 54/60 (90%) loci. However, there was discordance in heterogeneity status in 40/58 genetic loci . Furthermore, there was loss of concordance in miRNA expression status between primary and metastatic tumours, and 57.14–80.95% of the primary-metastases tumour pairs showed altered primary-metastasis relative expression in all the miRNAs tested. Moreover, 16 of 20 (80%) tumour pairs showed alteration in at least 3 of 6 (50%) of the protein biomarker pathways analysed.Among the primary and metastatic tumours the mutation rates for the individual genes are as follows:The molecular alterations of primary colorectal tumours differ significantly from those of their matched metastases. These differences have profound implications for patients’ prognoses and response to therapy.The online version contains supplementary material available at 10.1186/s12935-023-03020-7. Though metastases are the main cause of colorectal cancer deaths, much more is known about the underlying molecular features of the primary disease than the more advanced disease stages. This is because only a relatively limited number of studies have performed on metastatic colorectal carcinomas [Although significant advances in adjuvant chemotherapy for colorectal cancer (CRC) were achieved in the past few decades, the 5 year survival rate for the disease is still poor and as such, more than half of all colorectal carcinoma patients are expected to die from metastasis within this period . Thus thrcinomas . Overwhercinomas , 5. Morercinomas . As a corcinomas . In genercinomas .Generally, multiple and consecutive genetic alterations are needed for cancer development and some patients may have coexistent alterations in two or more different signalling pathways , 10. CRCAdditionally, the characterization of oncogenic mutations in metastatic disease could represent potential therapeutic targets. However, obtaining biopsies from metastatic sites for molecular testing involves an invasive procedure. Therefore, patients and clinicians still prefer using the least invasive method possible for genetic testing. Recently, there has been a growing evidence suggesting that there exists inter- and intra-tumour genetic heterogeneity in a range of solid tumours including CRC, thereby raising the concerns that molecular profiling of primary tumours may not be representative of metastatic disease , 13. ComIn this study, we investigated the molecular discrepancies between primary colorectal cancer (pCRC) samples and their matched metastases to assess the mutation profiles of 22 primary-metastasis CRC pairs in 26 cancer-associated genes using Next-generation sequencing (NGS) . FurtherFor this study, 22 pairs of pCRC and metastasis were selected. All patients had undergone surgery between 2004 and 2005 at the Queen`s Medical Centre (QMC) Nottingham, UK. Moreover, the candidate tumour blocks were selected based on the availability of clinicopathological data. Tumour cellularity, i.e. the proportion of tissue sections with cancer cells, was determined in the primary and metastatic CRC samples by a trained pathologist. The clinicopathological features of the cases included in this study is shown in Additional file We previously reviewed the haematoxylin and eosin stained slides and performed DNA extraction . Total RTrusight™ tumour kit  which provides a comprehensive view of somatic variation in 82 exons of 26 genes involved in solid tumours and total length capture of 21.6 Kb. Genomic DNA from CRCs (primary and metastasis) FFPE was fragmented to 300 -330 bp segments. The primers, adapters and indexes were then ligated to the DNA fragments to construct libraries. The DNA fragments were hybridized and after enrichment, transferred into the template position in the MiSeq reagent cartridge and sequenced on the Illumina MiSeq sequencing platform using MiSeq reagent kits v2 as recommended by Illumina. The detailed procedure is in the Additional file In order to validate the mutations detected by NGS, the samples were also analysed using the quick multiplex consensus-PCR (QMC-PCR) in conjunction with a high resolution melting (HRM) protocol as previously described .−ΔΔct, using the tumour cellularity-normalised ΔΔCt. Relative fold change of ≥ 2 (i.e. 0.5 ≤ relative ratios ≥ 2.0) between primary/metastatic tumours and normal tissue wastaken as the cut-off.After creating cDNA using miScript II RT kit , the selected miRNAs were quantified using a miScript SYBR Green PCR kit (Qiagen) in a 7500 Fast Real-Time PCR System (Applied Biosystems). The miRNA-specific primer sequences and primer efficiencies obtained from assay optimization are listed in Additional file The TC-normalisation method is based on two assumptions: (i) any difference in ΔΔCt between tumour and normal tissues is caused by the proportion of the tumour sample containing tumour cells; (ii) all tumour cells are expressing the miRNA at the same levels.For protein extraction, 20 μm thick sections were macro-dissected from formalin-fixed paraffin-embedded (FFPE) tumour blocks to enrich for the tumour content. Then protein lysates were quantified and an equal amount of lysates were loaded into a 384-well plate and visualised using the 700 nm (red) channel on an Odyssey high-resolution scanner  at 21 μm resolution. For comparative analysis, the intensity signals from total protein was used to normalize signals generated from other biomarkers . Completwww.sdmproject.com/utilities/?show=FDR). P values and adjusted P values (for multiple testing) of < 0.05 were considered to be statistically significant.All calculations, unless otherwise stated, were performed in Excel spreadsheet version 2010.The Benjamini and Hochberg correction was applied to multiple testing at a false discovery rate of 0.05 (5%) using the online FDR calculator software  present in a subpopulation of tumour cells ), (ii) present in all tumour cells ), and (iii) present in a background of copy number variation ) [change in the background heterogeneity state in the metastatic tumour counterparts: any HS difference of ≥ 0.5 between the tumour pairs was regarded as significant change   module in the MEDCALC easy-to-use statistical software at https://www.medcalc.org/calc/test_one_proportion.php. Differences in proportions were considered significant at a P value of < 0.05.Using the formula mutant allele frequency (MAF) X 2/ tumour cellularity, the heterogeneity scores (HS) were determined for individual mutations found in primary and metastatic CRC as previously described –25. Then (1.05)) –25. AlsoIn addition, the mutant allele frequency ratios (MAFRs) were calculated relative to trunk and branch driver mutations as previously described . Then, thttps://www.graphpad.com/quickcalcs/kappa2/).Tumour cellularity-normalised microRNA expression levels in the primary and metastatic tumours were categorized into high, normal and low on the basis of their relative expression ratios, i.e. relative ratios (RR) ≥ 2 = high expression, RR between 0.5 and 2 = normal expression, and RR ≤ 0.5 = low expression. Then the concordance in miRNA expression between primary and metastatic tumours was sought using QuickCalcs Kappa calculator at  was determined using the ratio of the relative expression of primary tumour to that of the metastatic tumour. The cut-off for miRNA PM-RE change (up-regulation and down-regulation) was ≥ 2 (i.e. ≤ 0.5 primary-metastases ratio ≥ 2)  x Em), where Em = relative protein biomarker expression in metastatic tumour, ∆TC = difference in tumour cellularity between primary and metastatic tumour, expressed in percentage. The relative expression of each biomarker in the metastatic tumour was considered to be significantly altered (up-regulated or down-regulated) if the difference in the relative biomarker expression between the primary and TC-normalised metastatic tumour values  was greater than the standard deviation of the biomarker expression for the entire tumour population, primary and metastatic tumours combined  biological pathways using the KEGG pathway analyses at _pathway : cellulaSomatic mutation profiling data was generated for all 22 tumour pairs. A total of 60 somatic non-synonymous mutations were found in 10 genes, comprising 51 specific types of mutations and 9 of which are recurrent. Of the 51 specific mutation types 15 are small indels, whilst 36 are single nucleotide variations (point mutations). All 9 recurrent mutation types are point mutations. Thirty-five of the single nucleotide mutations have previously been described, whilst one is a novel mutation (APC c.3871C > T). The 15 small indels are comprised of 9 deletions and 6 insertion (including 2 duplications). All indels, made up of 6 known and 9 novel mutations, generated a frame-shift , APC (14 cases), and KRAS (8 cases) (see in Table TP53 (19 cases), APC (14 cases) and KRAS (8 cases). When the two tumour groups were combined, mutations were most frequently observed in TP53 (86%), APC (44%), KRAS (36%), PIK3CA (9%), SMAD4 (9%), NRAS (9%) and 4% for FBXW7, BRAF, GNAS and CDH1. However, for some cases multiple mutations were detected for a single gene. In total, 23 mutations were detected for TP53, APC (18), KRAS (10), PIK3CA (2), SMAD4 (2), NRAS (2) and 1 mutation for FBXW7, BRAF, GNAS and CDH1.In primary CRC cases, the median number of mutations per tumour was 2.63 (range = 0–7) over a median of 2.27 genes (range = 0–4). The matching metastases had a median of 2.54 mutations per tumour (range = 0–5) over a median of 2.27 genes (range = 0–4). The most commonly mutated genes in primary CRC cases include TP53 9 cases, A total of 58 non-synonymous somatic variations in 10 genes were found in 22 primary tumours whereas 56 were found in metastatic cases. The mutant allele frequency was 1.03-fold higher in primary CRC than metastatic CRC. Four mutations in primary tumours were not seen in the metastatic tumours (private for primary) whilst two mutations present in the metastases were not seen in the primary tumours (private for metastasis). Although the paired primary and metastatic CRCs were not 100% identical, a Pearson’s correlation test shows no significant differences between them (p > 0.05).KRAS (G12V) and TP53 (H179R) whereas the metastatic tumour was wild-type at these loci. This case also had mutations in APC, SMAD4, KRAS (Q61H) and TP53 (V175A) which were identical in primary and metastasis. In case number 2, the primary tumour had an exon 8 GNAS mutation whereas the metastatic tumour was wild type. This case also had mutations for APC, KRAS and SMAD4 which were identical in the primary and metastatic tumours. In case number 3, mutations were found for NRAS (Q61K) and TP53 (I254N) in the metastatic tumour whereas the primary was wild-type. This case also had APC and TP53 (G266E) mutations which were identical in the primary and metastasis. The primary tumour for case number 4 contained an exon 8 mutation in CDH1 whereas in metastasis this gene was wild-type. KRAS and TP53 mutations were identical this tumour pair.Discrepancies between the primary and metastatic tumours were seen in four cases. As shown for case 1 in Table 2 = 0.081, P = 0.030) , branching driver (TP53) and other  mutations, only the RAS-BRAF somatic mutations showed significant but poor correlation between primary and metastatic tumours  .The comparison of the primary and metastatic tumours showed poor correlation in the heterogeneity scores for the somatic mutations  = 10/58, STC/WT = 3/58, CNV/STC = 4/58, CNV/ATC = 1/58, CNV/WT = 1/58, and ATC/CNV = 3/58. In addition, there was an intra-status change in the heterogeneity states in 19/58 alleles, comprising an increase in heterogeneity score in the CNV/CNV background in 8/58 alleles, a reduction in HS in CNV/CNV background in 10/58 alleles, and an increase in heterogeneity score in STC/STC background in 1/58 allele. No inter- or intra-status change in heterogeneity was seen in 17/58 (29.3%) alleles between primary and metastatic tumour pairs. In total, 41/58 mutant alleles (70.7%) showed discordance in the background heterogeneity states between primary and metastatic tumour pairs . In comparison the MAFR between resection samples (Rx) and their paired diagnostic biopsies (Bx) (see reference 26) showed a high correlation  ; miRNA29a ; miRNA31 ; miRNA92  and miRNA224   . Whole protein lysates from 20/22 primary CRCs and their matched metastatic tumours were obtained and analysedfor the expression of 22 different proteins by reverse phase protein array. The samples were run in triplicates and the mean of these replicates for each target in each sample was calculated  between proteins and their isoforms/modified forms and between proteins that are closely related functionally  recommends testing either the primary tumour or the metastatic lesion based on the results ofseveral studies that highlighted high concordance (> 95%) of tastases , 31. Simtastases , 33.KRAS status respond to anti-EGFR therapy while others do not [KRAS mutation status if such mutations were present in only some tumour cells in one patient, but occur in all the tumour cells, or in a copy number variation background, in the other patient. Likewise, if the background status of predictive markers differs between primary and metastatic tumours the response to therapy in the cells in the primary site can differ than those in the metastatic one [However, it must be noted that patients’ prognoses and therapeutic responses depend on other factors apart from the presence of one somatic mutation type or the other. For example, intra-tumour heterogeneity—both spatial and temporal—is emerging as a relevant factor in therapy response and prognosis , 33–36. s do not –39. To patic one –39. In tchanges in the metastatic tumour are important in therapy response [Differential mutant allele frequencies can therefore indicate differential prognosis and response to therapy. Therefore, actual mutant allele frequencies may be more informative than the currently adopted present-or-absent mutation approach, and may find more clinical importance in the near future. However, any method of MAF assessment must take into cognisance the diluting effect of ‘contaminating’ stromal DNA and tumour cellularity on the measurement of MAF. In this study we used the mutant allele frequency ratios (MAFRs) to compare the MAFs between primary and metastatic tumours, as previously explained [Furthermore, recent studies have demonstrated that the mutant allele frequency (MAF) response . Many inresponse –42. Diffxplained , and fouMicroRNAs are small single-stranded RNAs of approximately 22 nucleotides in length. They are involved in gene regulation and function by down-regulating gene expression via the inhibition of mRNA expression or via the promotion of target RNA degradation . ThroughDuring tumour progression gene expression patterns change and this is consequent upon changes in gene regulatory patterns . WithoutAlthough the miRNA profiles have been used—at least at clinical research level—in the prognostication of CRC patients , 57–59, To analyse our RPPA data, we developed a novel approach which utilizes the standard deviation as the cut-off for adjudging changes in expression levels between primary tumours and their matched metastases, and validated the approach by comparing the alteration patterns found for proteins and their isoforms, as well as for proteins which are functionally related. We reasoned that if our approach was valid, functionally related proteins and protein isoforms should show similar alteration patterns. Having validated our approach, we proceeded to analyse the protein biomarker expression data using a stringent biological pathway analysis approach. We used the alteration of 50% of the pathways analysed as the cut-off for significant alteration of overall expression profile between primary tumours and their matched metastases, since alteration in any one pathway in biological systems is associated with perturbation of (an)other pathway(s) . We founDifferential expression of protein and miRNA markers between primary tumours and their matched metastases is evidence of temporal heterogeneity of CRC , 34–36. In summary, we have evaluated primary CRCs and their matched metastases at the genomic, transcriptomic and proteomic levels, and found that primary tumours are inherently different from their corresponding metastases. These findings have both prognostic and therapeutic implications for CRC patients.Additional file 1. Data_biomarker pathways.Additional file 2. Data_general.Additional file 3. Data_miRNA.Additional file 4. Data_RPPA markers.Additional file 5. Data_somatic mutation profile.Additional file 6. Fig. S1. High Resolution Melting Difference Curve showing aberrant melting at APC exon 15 for samples 3, 5, 8, 9 and 12.Additional file 7. Fig. S2. Heat-map showing different signalling pathway intermediates studied in primary CRC (22) and metastasis (22) using RPPA. Rows represent the different signalling molecules studied. Green and red denote markers that are present at low levelsAdditional file 8. NGS.


Pancreatic cancer (PC) is known to be difficult to treat, even with standard-of-care chemotherapies. One emerging option for treating PC involves high-dose radiation therapy as a means to help control the growth and even destroy cancer cells. However, the pancreas is surrounded by many gastrointestinal organs and blood vessels; therefore, the implantation of a gold fiducial marker can help precisely target the pancreatic tumor. Our aim was to investigate whether implantation of fiducial markers through endoscopic ultrasound guidance is safe and feasible and to also document the radiation characteristics of this emerging high-dose radiation treatment option. n = 82) PC patients referred for EUS-guided fiducial placement by a single endosonographer at a tertiary cancer center. Data regarding EUS-related technical details, SBRT characteristics, adverse events, and continuous visibility of fiducials were recorded and analyzed. Most patients included in the study had either locally advanced disease  or borderline resectable disease . Eighty-two PC patients underwent the placement of 230 fiducial markers under EUS guidance. The technical success rate of the fiducial placement was 98%. No immediate EUS-related adverse events were reported. The average time to the simulation CT after fiducial placement was 3.1 days. Of the 216 fiducial markers used for the SBRT delivery, 202 fiducial markers were visible on both the simulation CT and the cone beam CT scan. A median dose of 40cGY was given to all the patients in five fractions. Of these, 41% of the patients reported no SBRT-related toxicities during the follow-up. Fatigue and nausea were the most reported SBRT-related toxicities, which were seen in 35% of the patients post-SBRT. Our results demonstrate that EUS-guided fiducial placement is safe and effective in target volume delineation, facilitating SBRT delivery in PC patients. Further clinical trials are needed to determine the SBRT-related survival benefits in patients with pancreatic cancer.Accurate delivery of stereotactic body radiotherapy (SBRT) to pancreatic tumors relies on successful EUS-guided placement of fiducial markers. The aim of this study is to report the technical feasibility and safety of EUS-guided fiducial placement and to evaluate the characteristics and technical benefit of SBRT in a cohort of patients with pancreatic cancer (PC). A retrospective chart review was performed for all ( Pancreatic ductal adenocarcinoma (PDAC) is considered one of the most lethal and therapeutically resistant malignancies, with a five-year survival rate of around 11% [The use of radiation therapy (RT) in the neoadjuvant, adjuvant, or palliative setting of gastrointestinal (GI) malignancies has been continuously changing. The data suggest that RT for PDAC patients in the neoadjuvant setting (resectable/borderline resectable disease) may increase local control and the likelihood of a negative margin resection ,3,4. In Standard radiation therapy doses for pancreatic tumors have failed to improve survival, due to the technical and anatomic limitations of the nearby GI organs. However, delivering a high dose of radiation to pancreatic lesions has been shown to have improved overall survival and recurrence-free survival. Thus, stereotactic body radiotherapy (SBRT), which delivers high-dose radiation, has become a new therapeutic option for treating pancreatic cancer .Image-guided radiotherapy (IGRT) and SBRT use advanced imaging technologies to verify and localize the target lesion during radiotherapy to decrease the treatment margins, thus decreasing toxicity to surrounding tissues. In order to safely deliver a higher dose of radiation to the pancreas while avoiding radiation toxicity to surrounding tissue, an accurate localization of the target tumor during respiration is necessary. Image-guided radiation therapy employs fiducial markers to precisely locate the target lesion during treatment and to monitor the tumor in real time, ensuring that radiation is administered with high precision. Fiducials are metallic markers, typically composed of gold or platinum, or liquid radiopaque markers that are strategically placed in close proximity to or within a target lesion. These markers serve as internal landmarks that facilitate the real-time tracking of the lesion. Most fiducial markers are gold, measuring 3 to 5 mm in length and about 1 mm in diameter, and have varying degrees of visibility in IGRT images . EndoscoAs the role of SBRT for palliative or curative intent for pancreatic cancer (PC) continues to emerge, EUS-guided delivery of fiducial markers will also become a more common procedure for endosonographers. Therefore, the aim of this study is to report the technical feasibility and safety of EUS-guided fiducial placement and to evaluate the technical feasibility and safety in consecutive patients with PDAC who have undergone SBRT at our institution since the launch of EUS-guided fiducial placement in 2016.This single-center retrospective study was approved by The University of Texas MD Anderson Cancer Center’s institutional review board. A retrospective chart review was performed for all PDAC patients referred for EUS-guided fiducial placement by a single endosonographer (M.S.B.) at a tertiary cancer center (The University of Texas MD Anderson Cancer Center) between 2016 and 2022. The data were retrospectively extracted from each patient’s electronic medical records and endoscopic database, which included patient demographics, indications for fiducial placement, fiducial marker deployment system properties, EUS-related technical details, procedure- and radiation-related adverse events, and SBRT dosing/planning.All the EUS procedures were performed using a linear-array echoendoscope  and the fiducials were deployed by either a commercial pre-loaded 22G fine needle aspiration (FNA) needle or were physically backloaded into standard 22G FNA needles. All the patients underwent a full EUS assessment prior to the fiducial placement in order to evaluate the tumor size and vasculatures surrounding the FNA needle trajectory. Once the tumor characteristics were identified, the FNA needle holding the fiducial marker would then be inserted into the target tumor using multiple EUS viewing planes. All the EUS procedures were given periprocedural IV antibiotics , and the patients were under deep sedation according to the institution’s standard anesthesia care guidelines. The continuous visibility of the fiducial markers was analyzed by an experienced radiation medical physicist on the simulation CT and during the final treatment fraction on the cone beam CT.p-value ≤ 0.05 was considered statistically significant. The distribution of continuous variables was summarized by means and standard deviations. The distribution of categorical variables was summarized using frequencies and percentages. The statistical analysis was carried out using SPSS Statistics software .A descriptive statistical analysis was performed. All the statistical analyses were two-sided, and a During the study period, a total of 82 PDAC patients underwent the placement of 230 fiducial markers under EUS guidance. The median age for the patient cohort was 69 years , with the majority of patients being male (56%) . Most ofOf the 230 fiducial markers  that were placed under EUS guidance, 166 (72%) fiducials were implanted using a backloaded technique, while 64 (28%) fiducials were implanted with a preloaded FNA needle . Gold AnTechnical success was defined as the capability to deploy the fiducial markers in the intended location. The technical success rate of the EUS-guided fiducial placement was 98% in the current study. Technical difficulties caused by intervening blood vessels in the FNA needle pathway were noted in two (2.4%) patients. A duodenal invasion was found during pre-fiducial deployment EUS evaluation in one case, and therefore the fiducial placement was canceled. All the patients received periprocedural antibiotics, and no immediate adverse events such as perforation, pancreatitis, bleeding, or infection relating to the EUS procedure were reported.The average time for the patients to transition from the fiducial placement to the simulation CT was 3.1 days . A total of 78 (95%) patients received the SBRT at our institution following the fiducial placement . The remOf the 216 fiducial markers used for the CT simulation and SBRT delivery, 202 (94%) fiducial markers were clearly visible on both the CT simulation a and conThe median radiation dosage for the SBRT in these patients was 40 Gy  over five fractions. Thirty-two patients (41%) reported no SBRT-related toxicities during follow-up with their radiation oncologist within 2 weeks following the SBRT. The most reported SBRT-related toxicities in this patient group were fatigue and nausea, which were seen in 27 patients (35%). Seventeen patients reported mild abdominal pain, and four patients reported nausea and vomiting post-SBRT. No serious adverse events were reported relating to the SBRT.SBRT has emerged as a promising treatment option in pancreatic cancer care in resectable/borderline resectable, locally advanced, or palliative settings. The placement of fiducial markers directly into the pancreatic tumor and/or tumor periphery under EUS guidance is useful for targeting purposes ,13. WhilThe first case series of EUS-guided fiducial placement in patients with abdominal and mediastinal malignancies was reported by Pishvaian and colleagues . Since tThe placement of the fiducials was determined by several factors, including patient anatomy and a multidisciplinary discussion between the endoscopist and radiation oncologist, as well as a discussion with the patient about EUS-guided placement of fiducials. Based on our current study results, the technical success rate of EUS-guided fiducial placement is very high (97%). Similarly, our previous meta-analysis of studies evaluating the technical aspects, safety, and efficacy of EUS fiducial placement in gastrointestinal malignancies, reporting on 1155 patients, revealed favorable results . The maiIn the current study, a total of 230 fiducial markers were deployed; 166 (72%) fiducials were implanted using a backloaded technique, while 64 (28%) fiducials were implanted with a preloaded FNA needle. A randomized controlled clinical trial including 44 patients with PDAC showed that EUS-guided placement of preloaded fiducial markers required less time but produced similar results compared with traditional backloaded fiducials . No statNo significant EUS-related adverse events were reported for the patients included in our study. The results are in line with our previous meta-analysis that yielded a low rate of adverse events (4%), indicating that EUS-guided fiducial placement is a safe procedure ,15,18,19According to our results, 94% of the fiducial markers were clearly visible on both the CT simulation and cone beam CT scan obtained on the last day of the SBRT delivery, which was evaluated by our institution’s radiation physicist team. Only 6% of the fiducials placed under EUS guidance were not useful for SBRT delivery, most likely due to migration or poor visibility. Spontaneous migration of fiducial markers can occur; however, no migration-related adverse events were reported in our study cohort. Similarly, none of the patients in our study underwent repeat EUS for placement of additional fiducials. Spontaneous migration of fiducial markers has been reported to be related to post-procedure inflammation resolution and from the tumor-shrinking caused by successful treatment ,24,27. TThe main goals of SBRT for the study cohort patients as determined by their GI radiation oncologist were either a consolidative intent by preventing disease progression and facilitating local control for patients with locally advanced PDAC, or a curative intent for patients with resectable  or borderline resectable PDAC. The use of SBRT has been correlated with enhanced local control and progression-free survival in comparison to conventional radiation therapy. This is attributed to the ability of SBRT to administer higher radiation doses while maintaining acceptable toxicity profiles ,29,30. AWith the advent of newer technologies that enable high-quality soft tissue imaging for IGRT, such as CT-on-rails or MRI-linac, fiducials may not be necessary . In caseThe limitations of this study should be noted. The results of this study are not a clear reflection of the outcomes of other hospitals, as this study was conducted retrospectively in a single tertiary cancer center. Furthermore, the results of this study should not be reflective of other anatomical sites where fiducial marker placement can be of benefit. The results may have had some minor complications omitted because there was a lag in follow-up evaluation between post-procedure recovery evaluation by the endoscopist and endoscopy staff and pre-CT simulation evaluation by the radiation oncologist. The use of three different types of fiducial markers may have skewed the visibility results, as the Medtronics and Lumicoil fiducial markers were bigger in diameter than the Gold Anchor fiducials that were used in the majority of patients. Furthermore, not standardizing the fiducial loading technique between backloading and pre-loading affected the overall length of the procedure and sedation time for all the patients, which could also affect the reporting of minor complications. It is important to note that these results should be replicated in a prospective clinical study to fully determine the objective measures of post-EUS complications, as well as radiation toxicities from SBRT targeting pancreatic lesions.Accurate and precise delivery of SBRT to a pancreatic lesion relies on the successful implantation of fiducial markers via EUS guidance. EUS-guided fiducial placement is a safe and feasible procedure with a measured success rate of 97% in patients with locally advanced or borderline resectable PDAC, according to our study. Furthermore, 97% of the patients successfully completed the SBRT. Although spontaneous migration of fiducial markers can occur, the rate of fiducial migration is relatively low (5%), and no migration-related complications occurred in our study. SBRT-related toxicities during follow-up were noted in many of our patients; with fatigue and nausea being the most commonly reported complaints. No serious adverse events were reported relating to the SBRT procedure. The main obstacle to achieving effective placement of fiducial markers is the intervening vasculature. In contrast to percutaneous and intraoperative methods, EUS-guided placement offers a less invasive alternative that facilitates precise access to deep anatomic structures located in the mediastinum, abdomen, pelvis, and retroperitoneum; however, comparative studies on this matter are lacking. The placement of fiducials is an additional application for interventional EUS that has the potential to broaden the indications for SBRT by enabling access to anatomical structures that may have been previously inaccessible. Further clinical trials are needed to determine the SBRT-related survival benefits in patients specifically with pancreatic cancer.


Pagiophloeus tsushimanus (Coleoptera: Curculionidae) larvae, Journal of Insect Science, Volume 23, Issue 2, March 2023, 14, https://doi.org/10.1093/jisesa/iead019This is a correction to: Heng Qiao and others, Isolation and characterization of gut bacteria associated with the degradation of host-specific terpenoids in Funding statement. This should read additionally: “[...]the Shanghai Landscaping & City Appearance Administrative Bureau (Grant number G161206), Postgraduate Research & Practice Innovation Program of Jiangsu Province (Grant number KYCX23_1236), and National natural science foundation of China (Grant number 32271884).” instead of “[...]and the Shanghai Landscaping & City Appearance Administrative Bureau (Grant number G161206).”In the originally published version of this manuscript, details were missing from the This error has been corrected in the article.


The new Cystic Fibrosis (CF) transmembrane conductance regulator modulator therapy combination  has significantly improved the quality of life of children with CF and decreased the rate of pulmonary exacerbations (PEx). The focus of our study was to examine the infection characteristics of PEx in children with CF before the availability of ETI (2018) and following ETI approval and during emergence from COVID-19 (2022).We conducted a multicenter (5 sites) retrospective review collecting data on children with CF over 12-month periods in 2018 and in 2022 who were hospitalized for PEx and treated with IV antibiotics. We analyzed the number of PEx, bacterial and fungal organisms identified by CF respiratory cultures and IV antibiotics used empirically at hospital admission.The number of hospitalizations for PEx decreased from 414 in 2018 to 163 in 2022. The most common organisms identified in all centers were Pseudomonas aeruginosa (Pa), Staphylococcus aureus (Sa) and Candida species, with no apparent overall difference in the relative percentages of pathogens isolated between 2018 and 2022 (figure 1), although fewer children were infected with methicillin-resistant S. aureus (MRSA) in 2022 (2.7%) than in 2018 (10%) (p=0.014). There was variability in the type of antipseudomonal agents and antistaphylococcal agents used empirically both within and between the different centers (figure 2).Pathogens isolated from respiratory culture associated with PEx demonstrated similar rates of isolation of pathogens in the 2 time periods, consistent with historical CF pathogen isolation. The decreased number of PEx in 2022 compared to 2018 is consistent with the widespread use of ETI. The variability of usage antipseudomonal agents between centers and between years of data collection requires further investigation.Andrea Hahn, MD, MS, TGV Dx: Advisor/Consultant Antonio C. Arrieta, MD, FIDSA, FPIDS, Astellas Pharma Global Development, Inc.: Advisor/Consultant|Astellas Pharma Global Development, Inc.: Grant/Research Support|Astellas Pharma Global Development, Inc.: Honoraria|Cumberland Pharmaceutical: Grant/Research Support|IDbyDNA: Advisor/Consultant|IDbyDNA: Grant/Research Support|Melinta: Grant/Research Support|Merck: Advisor/Consultant|Merck: Grant/Research Support|Nabriva: Grant/Research Support|Paratek Pharmaceuticals: Grant/Research Support|Pfizer, Inc: Advisor/Consultant|Pfizer, Inc: Grant/Research Support|Roche/Genentech: Grant/Research Support|The Medicine Company: Grant/Research Support


Of the 18 cases, five patients were diagnosed with acute meningitis, three with acute meningitis combined with facial neuritis, three with acute meningitis combined with polycranial neuritis, and the remaining seven with various clinical diagnoses. Typical clinical symptoms included headache (15), fever (9), and rash (11). Cranial or spinal MRI showed abnormalities in 12 patients, and 17 patients had obvious neurological symptoms. The predominant genotype of VZV in this study was genotype J . All patients were treated with acyclovir/penciclovir and dexamethasone, 16 recovered and 2 died. Our study highlights the good performance of mNGS in diagnosing CNS infection caused by VZV. It could provide additional diagnostic evidence in patients with diverse clinical spectrum and variable manifestations.Clinical presentation of central nervous system (CNS) infections caused by varicella-zoster virus (VZV) is highly sophisticated, making identification challenging. We retrospectively reported 18 cases of VZV neurologic disease confirmed by metagenomic next-generation sequencing (mNGS). The detection rate of mNGS was higher than that of PCR assay (100 vs 66.7%,  SpontanThe diagnosis of central nervous system (CNS) infection caused by VZV is usually based on the temporal association of the rash with onset, the synthesis of anti-VZV antibodies, and the presence of VZV DNA in cerebrospinal fluid (CSF) . SeveralAs a target-independent method, metagenomic next-generation sequencing (mNGS) enables simultaneous and unbiased detection of almost all pathogens from clinical samples , which h22.16/L, CSF cytology analysis showing lymphocytic inflammation, lesions by brain imaging indicating encephalitis, and EEG findings suggesting encephalitis; (3) positive nucleic acid for VZV (PCR or mNGS), or positive CSF and/or serum IgM antibodies. Cases diagnosed with other etiologies were excluded [This study retrospectively reviewed clinical data of patients with VZV neurologic diseases, who admitted to our hospital from March 2021 to July 2022. All enrolled patients met the following inclusion criteria: (1) any alteration in mental state  or the presence of seizures; (2) at least one of the following symptoms: body temperature ≥38℃ (within 72 h before or after onset), focal neurological manifestations, CSF white blood cell count >5 × 10excluded .For each case, clinical records of prodromal symptoms, cranial or spinal imaging findings, biochemical tests, laboratory tests, and the clinical course of the disease were investigated. Additional records including treatment strategies, clinical outcomes, and relevant follow-up data were also collected.Ethics statement: The studies involving human participants were reviewed and approved by the Ethical Review Committee of The Affiliated Ganzhou Hospital of Nanchang University. The participants provided their written informed consent to participate in this study. Written informed consent for minor participants was provided by either parent or guardians of participants.2.2ESR-104G (VZV-IgG) and ESR-104M (VZV-IgM)  were used for IgG and IgM antibody measurement. There exist three distinct threshold levels of serum IgM  that serve as the basis for positive judgment. A level of serum IgM below 0.470 is deemed negative, while a level between 0.470 and 0.653 is considered critical. A level between 0.653 and 0.7583 is classified as weakly positive, and a level greater than 0.7583 is deemed positive. Similarly, there are also three threshold levels of serum IgG  that serve as the criteria for positive judgment. A level of serum IgG below 0.198 is classified as negative, while a level between 0.198 and 0.369 is considered critical. A level between 0.369 and 0.469 is classified as weakly positive, and a level greater than 0.469 is deemed positive.ORF62 gene was amplified, the primers were 5-GGAGACCAGAGAGGGTCACT-3 and 5-AACCCTCTAGGCCGATCTGT-3. The PCR product was confirmed by gel electrophoresis.PCR was utilized for VZV nucleic acid detection. DNA was extracted from CSF samples using DNA Extraction Mini Kit . All amplifications were performed using SU PCR Mix  and in Eppendorf Mastercycler Gradient PCR Thermal Cycler . In the PCR reaction, 117-bp fragment of 2.3CSF samples were collected from patients and stored at −20℃. The samples were sent for mNGS detection . DNA was extracted and purified following the standard procedures of QIAamp DNA Micro Kit . Qubit 4.0  was used for DNA concentration and quality control. Libraries were constructed using QIAseq Ultralow Input Library Kit . After qualified, libraries were sequenced on Nextseq 550 platform .2.4fttp://ftp.ncbi.nlm.nih.gov/genomes/). Genotype analysis was performed based on metagenomic data. For VZV, if the number of VZV-specific sequence is greater than or equal to three and not detected in the no template control, it will be judged as mNGS positive.The generated raw reads after mNGS sequencing were filtered by removing adapters, low-quality and short reads (<35 bp), and the clean data were obtained. Bowtie2 was used for excluding human sequences by mapping the clean data to the human reference genome (hg38). For microorganism identification, the remaining reads were then aligned to the published microbial genome databases, which were downloaded from National Center for Biotechnology Information  and eight females (44.4%). The ages of these patients ranged from 15 to 85 years old, with a median age of 57 years old. Of the 18 patients in our study, five patients were diagnosed with acute meningitis, three with acute meningitis combined with facial neuritis, three with acute meningitis combined with polycranial neuritis, one with acute meningitis combined with myelitis, choriomeningitis, and myeloradiculitis, one with acute meningoencephalitis, one with acute meningoencephalitis combined with bilateral facial neuritis, one with acute meningoencephalitis combined with polycranial neuritis, one with acute meningoencephalitis combined with conjunctivitis and blepharitis, one with acute brainstem encephalitis complicated with myelitis, and one with acute choriomeningitis and myelitis. Typical clinical symptoms included headache (15), fever (9), and rash (11), another two patients developed fever after pulmonary or other organ infection. In addition, ten patients had underlying diseases including hypertension (3), diabetes (3), autoimmunity disease (2), cerebrovascular disease (1), and beta-thalassemia (1). The CSF leukocyte counts were remarkably elevated in 13 patients, revealing lymphocyte predominance, and the CSF protein levels increased in 13 patients. Twelve patients had abnormal brain or spinal cord MRI, and 17 patients had obvious neurological symptoms. The clinical characteristics of all subjects are listed in 3.2mNGS and PCR were performed on all 18 CSF samples, while serum VZV IgG and IgM antibodies were examined in 16 patients. According to the mNGS detection, VZV unique reads were detected in all cases with specific sequences ranging from 3 to 259,789, the relative abundance ratio of VZV ranged from 0.03 to 99.99%, and its coverage ranged from 0.18 to 99.95%. Ten cases were with more than 90% VZV coverage by mNGS detection. Furthermore, genotype J was identified in all these ten cases, indicating genotype J as the major genotype of VZV in this study.p < 0.05) and serum IgM antibody  measurement. All patients were treated with acyclovir/penciclovir and dexamethasone. Two cases died, the others were discharged and fully recovered after 1-month follow-up. The yield of different etiological examinations, therapy, and prognosis of all patients are summarized in VZV IgG antibodies were present in serum samples from 15  patients, and VZV IgM antibodies were observed in 11  patients. Twelve  patients had positive PCR results for VZV in CSF. The detection rate of mNGS for VZV was higher than that of PCR assay . Thoracic and lumbar spine MRI enhanced scans are shown in Case 1, a female patient aged 67, was admitted to the hospital on December 27, 2021, with generalized body ache and marked weakness in both lower extremities for 10 days. She developed pain in both lower limbs without obvious inducement on December 7, 2021, mainly in below knee with upper back dull pain and weakness in both of her lower limbs. She was treated at a native Hospital on December 12, 2021. On the next day, she developed herpes in the sacrococcygeal region, with significant pain in the lower back and progressive radiating pain in the bilateral lower extremities. She was transferred to the emergency department of our hospital because of the gradual worsening of her condition . The patient was preliminary diagnosed with acute myelitis. Routine blood examination reported that percentage of neutrophils (82.20%) and C-reactive protein (4.50 mg/L) were elevated. A lumbar puncture was done on December 29, 2021; the results showed an elevation in protein (95.19 mg/dL) and a normal CSF pressure (175 mmH3) and a total protein of 144.7 mg/dL. Brain MRI showed multiple nodules in the bilateral frontotemporal and parietal lobes, left insula, and bilateral cerebellum hemispheres  and glucose level (6.39 mmol/L). He was diagnosed with brainstem encephalitis and myelitis, and was treated with intravenous injection of acyclovir and dexamethasone. On April 15, 2022, the patient developed fever, sputum production, and rales on auscultation of the lungs. Aspiration pneumonia was considered, and the patient was given piperacillin/tazobactam . On April 17, 2022, CSF mNGS revealed 12527 VZV specific reads. ECG showed sinus bradycardia, and a temporary pacemaker was inserted to assist the rhythm. Despite aggressive resuscitation, the patient failed to breathe by himself, his family abandoned treatment on April 22, 2022.Case 14, a male patient aged 47, was admitted to the hospital on April 14, 2022, with neck pain for 10 days and limb weakness for 2 days. The patient had no specific medical history. On April 4, 2022, he woke up with neck pain without any obvious inducements and then developed a scattered rash all over his body the next day. On April 12, 2022, the patient presented to a local hospital with weakness in his extremities, which was later considered as myelitis . He had Case 15, a female patient aged 77, was admitted on April 4, 2022, with recurrent dizziness for half a month, aggravated left eyelid opacification, and orofacial distortion for 8 days. She suffered from dizziness and nonprojectile vomiting after catching a cold on March 20, 2022. She was hospitalized in our emergency department and was diagnosed with benign paroxysmal positional vertigo. Her symptoms improved and was discharged on March 27, 2022. After discharge, she still presented with incomplete closure of the left eye and mouth angle askew to the right side, and the discomfort and dizziness continued. The patient was readmitted after self-administered proprietary Chinese medicine, which had no significant effect. The patient was diagnosed with Ramsey-Hunt syndrome. She was treated with pitavastatin and promethazine for dizziness control, dexamethasone sodium phosphate for anti-inflammatory, acyclovir for anti-viral treatment, vitamin B1, and mecobalamin for neuronutrition, but she still felt dizziness. On April 5, 2022, the patient experienced dysphagia and was unable to ingest much food, but her family rejected nasogastric tube feeding. On April 12, 2022, the patient presented with neck stiffness and positive meningeal irritation signs. On April 13, 2022, CSF analyses demonstrated elevated protein levels (58.54 mg/dL) and an elevated glucose level (6.39 mmol/L). On April 14, 2022, the patient had loss of consciousness, cold extremities, low blood pressure, increased heart rate, tachypnoea, and shock. The PCT, routine hemogram, coagulation factor level, serum electrolytes, kidney and liver function of this patient were also abnormal. Cardiac color Doppler ultrasonography showed an atrial septal defect. The patient was diagnosed with septic shock and atrial septal defect, biapenem combined with linezolid was added for antiviral treatment. On April 15, 2022, the patient presented with right-sided extremity weakness, her brain MRI revealed acute infarction of cerebellar hemispheres, frontal lobe, basal ganglia, corona radiata, the right basal ganglia, and multiple spot shadows in the lateral ventricles . In the 5The crucial diagnostic sign of VZV CNS infections is the presence of specific viral DNA in the CSF or blood. In this study, we described 18 patients with CNS infection caused by VZV. The advantages of mNGS over traditional microbiological assays for diagnosis of VZV were highlighted, consistent with a previous study . Most ofVaricella is usually the primary infection symptom of VZV, and VZV is latent in all neuraxis-wide ganglionic neurons . VZV is As a typical clinical feature of VZV infection, the rash may be absent in many cases. The absence of rash increases the difficulty of diagnosis. In this study, there were seven (38.9%) VZV patients who exhibited no rash. For instance, patient 9 presented with headache, without fever or rash as initial symptoms. The initial diagnosis of the patient was meningoencephalitis, but he developed bilateral cranial neuritis and then secondary to cerebral vasculitis. He had an elaborate spectrum of clinical presentations and complex diagnosis. Due to the diversity of clinical manifestations, the clinical diagnosis of VZV can be difficult, especially in the absence of varicella. The introduction of mNGS confirmed VZV infection in this case. This case indicated that bilateral cranial neuritis caused by VZV can be an early clinical symptom, as well as a complication of recovery period. Comparing with PCR and IgM test, mNGS revealed significant advantages in diagnosing VZV infections, especially in the absence of a rash. For example, one patient who developed a rare asymptomatic infection-induced paraplegia after anti-retroviral therapy, was confirmed as VZV-induced thoracic myelomyelitis by using CSF mNGS . Fang etDrugs approved for the treatment of VZV-associated disease include acyclovir, its oral prodrug valacyclovir, and famciclovir, a penciclovir prodrug . Short cThis study also has some limitations. This is a single-center retrospective study, and some clinical data in our cases were incomplete, which may reduce its clinical significance. The number of cases in this study is relatively small and there is a lack of controls for healthy individuals or cases of CNS infection with other pathogens. Further prospective research with a larger sample size is warranted.6In the article, we represent 18 cases of VZV CNS infection, which demonstrate different clinical manifestations and various imaging characteristics. The diagnosis in some cases is difficult and may lead to death if delayed. mNGS allows an effective and rapid diagnosis for viral infection.


Yolk sac tumour frequently arises in the gonads as a type of germ cell tumour, though rare is a highly malignant ovarian tumour in children and prompt treatment should be done. We hereby report a case of malignant ovarian tumour presenting with an abdominal lump and increased urinary frequency. Different diagnostic modalities were used such as ultrasonography of the whole abdomen, contrast-enhanced computed tomography abdomen pelvis and tumour markers of betahuman chorionic gonadotropin and alpha-fetoprotein. This revealed an 18.2×14.3×10 cm mass likely a neoplastic germ cell tumour with minimal ascites. A tumour mass was found to arise from the left ovary and complete excision of the tumour along the left fallopian tube was done. Adjuvant chemotherapy started immediately. We hereby present a case of a 9-year-old girl with a huge yolk sac tumour of the left ovary which is rare in our setting and is presented here to differentiate any ovarian mass in this age group. Tumour markers for YST are α-FP and β-HCG.4 Schiller-Duval body, pathognomonic for yolk sac tumours, appears like a glomerulus in structure with a fibrovascular core.2Ovarian masses can be solid or cystic with multivariate histology and are rare in the pediatric population. Incidence of ovarian tumours increases with age, from 0.43 in 100,000 cases at 1 year age to 152 in 100,000 cases in 35-year-old patients.A 9-year-old girl was first presented to her general practitioner in local clinics with a history of fever with a maximum temperature of 38.33° C and was treated with some antibiotics on an outpatient basis. When the patient was assuming the supine position at her home, her mother noticed a bulge on her abdomen which upon palpation felt like a hard mass which was never talked about or examined. The patient was then brought to a tertiary care centre for further evaluation. The patient also complained of increased urinary frequency for 3-4 months, with no history of any vaginal bleeding or discharge. The patient's menarche had not started yet. The patient also complains of a recent loss of 3 kgs of weight in the past 3 months and night sweats a month prior. There was no history of any radiation exposure when she was conceived; the post-delivery status of the child was unremarkable. There was a history of bilateral ovarian cyst surgery in the mother of the patient. Furthermore, there is a history of ovarian cyst excision on the paternal side of the family (3 sisters of the father).On physical examination, a palpable, firm, non-mobile hypogastric mass was observed. USG showed a huge well-defined heterogeneous solid cystic lesion measuring 17.1x12.2 cm in the pelvic cavity possibly arising from the left adnexal region showing internal vascularity, possibly neoplastic in nature.Computed tomography revealed a large solid cystic mass measuring approximately 18.2x14.3x10 cm in the pelvis, lower and central abdomen with no calcification or fat densities. Solid components showed intense heterogeneous enhancement in the post-contrast study and peripheral wall enhancement in cystic components. Mass supplied by the branches of internal iliac arteries and predominant drainage into the left gonadal vein which is enlarged measuring 10 mm. Mild indentation into the superior surface of the urinary bladder is seen with mild compression of both ureters with mild dilation of both pelvicalyceal systems. The left ovary is not separately visualized with the normal uterus and right ovary. Mildly enlarged aortocaval and para-aortic lymph nodes measuring 16x11 mm and 13x10 m respectively. Minimal free fluid is seen in the peritoneal cavity .Features suggestive of malignant germ cell tumour arising from left ovary. The value of tumour markers, total beta-human chorionic gonadotropin and lactate dehydrogenase (LDH) were <2.39 mIU/ml and 417 U/l respectively and alpha-fetoprotein (AFP): 500 ng/ml. Thus, the patient underwent complete excision of the mass (weighing 3 kg and 40 g) along with the removal of the left fallopian tube .A gross examination of the specimen showed no breach in the capsule and cut section showing solid (grey-white) and cystic (showing mucin in space) areas with areas of haemorrhage and necrosis. Hematoxylin and eosin-stained sections of the specimen revealed tumour cells arranged in nests, sheets, glands and microcystic patterns. Schiller-Duval bodies were also seen. The findings were consistent with YST without lymphovascular, ovarian surface or fallopian tube invasion.The patient was discharged 4 days after the operation with unremarkable recovery. It was a staged I yolk sac tumour. The case was referred to a pediatric oncologist where she was advised to chemotherapy. The patient underwent four cycles of adjuvant chemotherapy and monitoring of chemotherapy effectiveness is done with an estimation of serum alpha-fetoprotein level.5 Young patients with ovarian tumours often complain of acute or chronic abdominal pain, vomiting, nausea, increase of abdominal volume or other symptoms induced by the compression on surrounding organs.2 However, our patient also first presented with fever and abdominal bulge but later stated the fact that she had prior different symptoms due to compression of the surrounding organs like frequency of urination.Yolk sac tumour which most frequently arises in the gonads as a type of germ cell tumour is rare in children but is highly malignant.6 and others too. In certain cases, abdominal-pelvic MRI may also be required. The ultrasound exam was very suggestive of the diagnosis in our case. As per other studies, AFP can be applied as a feasible tumour marker because its level was seen to be elevated in >90% of YST.7 In our case, AFP was found to be 500 ng/ml. The prognosis of patients can be monitored by the AFP level after the operation.7 It has been reported that the lowering level of post-operative serum AFP could be a useful marker for determining if residual cancer cells still exist after surgery. Serum alphafetoprotein has been used as a marker for the effect of postoperative radiation therapy and/or chemotherapy in cases of ovarian endodermal sinus tumours.6 AFP is specific in the yolk sac tumour, but it is not sensitive  because it can be seen in other cancers like hepatocellular carcinoma.The imaging tools are extremely useful in establishing the diagnosis of ovarian tumours and they usually consist of pelvic and abdominal ultrasounds, CT scans and MRIs. Ultrasound is widely used in the assessment of multiple pediatric pathologies, such as abdominal tumours,10The general treatment for YST is surgery for eliminating the primary tumour without severe morbidity. The surgical approach of adnexal masses can consist of either laparotomy or laparoscopy depending on factors related to both the tumour and the patient.10In our case, due to the huge size of the tumour, a laparotomy with complete excision was done and we did not encounter any post-surgical complications. The postoperative period was uneventful. Given the tumour size in our case, complete excision of the tumour along with a left fallopian tube and left ovary was done. For most of the patients with OGCT, unilateral scalping-oophorectomy with preservation of the contralateral ovary and the uterus was appropriate.11 After treatment, follow-ups are required such as abdominal and pelvic examination, CT, chest X-ray and AFP levels.As per guidelines, the present-day treatment for the yolk sac tumour is surgery and chemotherapy. Thus, the involvement of an oncologist is necessary. In the case of yolk sac tumours, most patients will need at least three cycles of chemotherapy. The bleomycin, etoposide and cisplatin (BEP) regimen is the best choice. A significant response to chemotherapy is indicated by a decrease in tumour marker levels following treatment. The diagnosis, the intervention, and the therapeutic plan must be based on the symptoms, pelvic ultrasound and potential serum markers. Furthermore, YST metastasizes through the hematogenous route in >50% of pediatric patients in comparison with only 4-6% of adult patients.Proper presurgical investigations are of major importance because the Proper orientation of the therapeutic plan, the surgical approach, the surgical team, and the type of surgical intervention is only possible through proper presurgical investigations. With the aid of the paediatrician's and gynaecologist's communication abilities, a successful relationship with the patient and parents should be developed to get the best outcomes.The clinical features of children with yolk sac tumours commonly present with abdominal pain and distension. However, our case presented initially with fever and later with abdominal distension and pressure symptoms due to the increasing size of the tumour. Management of the case was done by complete excision of the tumour along with the removal of the left fallopian tube and adjuvant chemotherapy. Due to the rarity of this tumour, it should be suspected in young children so that treatment can start early before metastasis. Prompt treatment and investigations should be started after the diagnosis of the tumour. A team effort is required for the best outcome in favour of the patient and parents.


MEIA urine diagnostic was developed as an enzyme immunoassay, by Johns Hopkins (JH) and Pearl Diagnostics. In a retrospective study of 310 people, the assay was 90.5% (95% CI 70-99) sensitive and 89.2% (95% CI 82-94) specific. We report results of a prospective study performed at JH Hospital (JHH).The MycoMEIA results in subjects with possible IA, mixed infections, or other infections (non-IA) were evaluated descriptively.People with suspected invasive aspergillosis (IA) were identified by screening of diagnostic tests sent by clinicians at JHH. Urines from consented subjects were tested in the clinical mycology lab within 8 hours of collection. Results were not used to inform care. Established diagnoses were adjudicated by clinicians blinded to lab results, using consensus criteria. Performance was analyzed in evaluable subjects who had CTs within 2 weeks of suspected IA. Findings supporting proven or probable IA, or no infection defined cases and controls, respectively. MycoMEIA urine tests.107 urines were tested from 72 subjects with suspected IA. 15 subjects had >1 samples tested. Subjects were mostly male , with median age of 51 (range 7 – 87 years). IA was suspected during treatment for heme malignancies , solid organ transplant (9), cancer (5), and rheumatologic disease (1). Of 72 subjects enrolled, 30 had possible IA (42%), 30 no infection (42%), 4 probable IA (6%), 4 mixed infections (6%) and 4 other infections, including bacterial abscess (1), fusariosis (1), fungal sinusitis (1) and otitis externa (1). The assay was 100% sensitive (95% CI 76–98) and 93% specific (95% CI 79–99) for IA, with likelihood ratio of 15. Five of 30 (16.7%) people with possible IA and 1 with fusariosis had positive MycoMEIA – Aspergillus test appears consistent with retrospective findings and favorable compared to current blood assays. The proportion of lab confirmed diagnoses using current commercial assays was low; high negative predictive value with urine testing may support restriction of empirical antifungals. Cross reactivity amongst pathogenic Ascomycetes is biologically anticipated and requires further study. The study remains ongoing.In prospective testing by the clinical lab, performance of the urine MycoNitipong Permpalung, MD, MPH, Alcimed: Advisor/Consultant|CareDx: Grant/Research Support|Cidara Therapeutics: Grant/Research Support|Clarion: Advisor/Consultant|ClearView: Advisor/Consultant|IMMY Diagnostics: Grant/Research Support|Merck: Grant/Research Support|Scynexis: Grant/Research Support Kieren Marr, MD, Cidara Therapeutics: Advisor/Consultant|Merck: Advisor/Consultant|Pearl Diagnostics: salary|Pearl Diagnostics: Ownership Interest|Sfunga Therapeutics: salary|Sfunga Therapeutics: Ownership Interest Irina Baburina, PhD, Pearl Diagnostics: salary Dana Neitzey, BS, Pearl Diagnostics: salary Patricia X. Marques, PhD, Pearl Diagnostics: salary Sean Zhang, MD, PhD, Applied BioCode: Grant/Research Support|IMMY Diagnostics: Grant/Research Support|KARIUS: Advisor/Consultant|Pearl Diagnostics: Grant/Research Support|Scanogen: Grant/Research Support|T2 biosystems: Advisor/Consultant|Vela Diagnostics: Grant/Research Support


As a vital parameter in living cells and tissues, the micro-environment is crucial for the living organisms. Significantly, organelles require proper micro-environment to achieve normal physiological processes, and the micro-environment in organelles can reflect the state of organelles in living cells. Moreover, some abnormal micro-environments in organelles are closely related to organelle dysfunction and disease development. So, visualizing and monitoring the variation of micro-environments in organelles is helpful for physiologists and pathologists to study the mechanisms of the relative diseases. Recently, a large variety of fluorescent probes was developed to study the micro-environments in living cells and tissues. However, the systematic and comprehensive reviews on the organelle micro-environment in living cells and tissues have rarely been published, which may hinder the research progress in the field of organic fluorescent probes. In this review, we will summarize the organic fluorescent probes for monitoring the microenvironment, such as viscosity, pH values, polarity, and temperature. Further, diverse organelles  about microenvironments will be displayed. In this process, the fluorescent probes about the “off-on” and ratiometric category (the diverse fluorescence emission) will be discussed. Moreover, the molecular designing, chemical synthesis, fluorescent mechanism, and the bio-applications of these organic fluorescent probes in cells and tissues will also be discussed. Significantly, the merits and defects of current microenvironment-sensitive probes are outlined and discussed, and the development tendency and challenges for this kind of probe are presented. In brief, this review mainly summarizes some typical examples and highlights the progress of organic fluorescent probes for monitoring micro-environments in living cells and tissues in recent research. We anticipate that this review will deepen the understanding of microenvironment in cells and tissues and facilitate the studies and development of physiology and pathology. Keeping the micro-environment healthy in cells and tissues is of vital importance to organisms. Multiple biological processes require a normal microenvironment, including viscosity, pH values, polarity, and temperature, to achieve basic life activity. As an important microenvironment, viscosity could control metabolic processes and diffusion-mediated processes at the cellular level, such as intra/intercellular signal transduction, the interactions of biomolecules, and proliferation of nitrogen species (RNS), etc. ,2,3. MorAs a vital constituent part in cells, organelles participate in various biological processes ,13. So, Over recent years, the probes used for visualizing organelle environment has aroused researchers’ attention. Therefore, a large quantity of fluorescence probes was developed to monitor the microenvironment in organelles. However, the comprehensive and critical reviews about the visualizations of microenvironment in organelles were quite rare. So, in this review, we systematically summarize the microenvironment-sensitive probes for imaging organelles. To grasp the topic, the review was categorized into four common classes  according to the diverse physical properties, and each class was divided into four membrane organelles  in Viscosity is a paramount cell microenvironment, which influences various cellular processes and interactions of biological molecules, such as signal transduction, protein aggregation, membrane fusion and so on ,23,24. TMitochondrial viscosity plays an important role in various life activities, such as the reflection of tricarboxylic cycles and respiratory state . MoreoveAs a typical fluorescence sensor of viscosity, molecular rotors could be manipulated by the molecular excited state planarization, and it was used to monitor mitochondria viscosity . As showIn view of the excellent properties of the fluorescent rotor, a mass of fluorescent probes sensitive to viscosity was developed. Park et al. have exploited a viscosity-sensitive mitochondrial fluorescence probe  . The visAs an important microenvironment parameter, lysosomal viscosity could reflect the status and function of lysosomes . So, lysIn view of the advantages of fluorescence technology, a lot of fluorescent probes were exploited to monitor lysosomal viscosity. Deng et al. exploited a viscosity-sensitive fluorescent probe (Lyso-V) for visualizing lysosome through linking BODIPY rotor with a morpholine moiety . The fluIn spite of the drawbacks of “off-on” probes stated above, the ratiometric fluorescent probes were exploited to visualize viscosity in dual colors. To achieve ratiometric visualization of lysosomal viscosity, Liu et al. have exploited a viscosity-responsive ratiometric two-photon probe (Lyso–Vis) with the through bond energy transfer (TBET) mechanism for tracking autophagy process . The lysAs the front barrier of the cell, the cell membrane controls many bio-molecular interactions through selective transportation of substances into cells . The celAlthough the probes stated above could visualize cell membrane viscosity, they could not quantify membrane viscosity. Thus, in the future, more probes could be developed to achieve the quantification of cell membrane viscosity, so as to provide effective guidance for viscosity-related diseases.Endoplasmic reticulum (ER) viscosity is one of vial parameters in cells and tissues, and the changes viscosity is caused by the accumulation of unfolded or misfolded proteins . Thus, iNowadays, the probes used for visualizing ER viscosity are usually designed by connecting rotor to ER targeting moiety. Kim et al. have exploited an ER membrane targeting probe (V-1) by linking BODIPY to coumarin with a long alkyl chain (n-C18) for monitoring ER viscosity . With thRecently, the viscosity changes of ER induced by ferroptosis has aroused researcher’s interest. Xiao et.al developed a new type of probe (L-Vis-1) by linking ER targetable group to a BODIPY rotor to sense viscosity with its fluorescence lifetime, and the real-time analysis of ER viscosity changes during different ferroptosis processes were achieved by L-Vis-1 and fluorescence lifetime imaging (FLIM) . Kim groIn addition, there are also some viscosity-sensitive probes for visualization of other organelles. Tian et al. have exploited two small organic molecules with optimized two-photon action cross-section. These probes are water-soluble, and they could ratiometric light up nucleic acid with two unique fluorescence . MoreoveAs a vital parameter in cells, the pH values are of vital importance to various life activities. Different organelles have discrepant pH values, such as mitochondria (pH~8), lysosome (pH:4.5–5.5), cytosol (pH:6.8–7.4) ,70. The The pH can reflect the states of various biological processes, including signaling, adenosine triphosphate production, cell cycle and death . Thus, vAs an acid organelle (acidic environment of 4.5–6.5) in living cells, the lysosome could accelerate the metabolism of macromolecular, and it is associated with human diseases and cell ageing . Thus, t+-induced ring opening, and it was used to track and detect lysosomal pH changes because of the rapid response, good reversibility, and high selectivity properties. Compared with the “off–on” type, the ratiometric probes were also exploited. Zhang group have developed a ratiometric fluorescent probe sensitive to pH for dual-channel imaging of lysosome by combining xanthane derivative and naphthalimide group . T. T93]. TRecently, single probes for visualizing two organelles in dual color were developed utilizing the discrimination of diverse organelles. Lin group have developed a pH-sensitive probe with dual color visualization of mitochondria and RNA, and the probe PVMR could target to mitochondria and release to apoptosis, due to the relationship properties between mitochondria and RNA . PVMR waAmong various microenvironments, cell polarity is a significant factor to reflect the state of cells, and cells also need to keep normal polarity to maintain specific functions . Most ceMitochondria are a kind of dynamic organelle for providing energy for cellular activities, and the polarity of mitochondria is a vital indicator for abnormal mitochondrial functions. Yin et al. have developed a probe sensitive to polarity with “off-on” properties, and they used it to study the polarity changes in mitophagy with a commercial autophagy tracker, MDC (dansylcadaverine) . MoreoveLysosome is a vital organelle with digestion function, and the polarity of lysosome is always changing . In many2O with diverse polarity  with lysosomal targeting. The probe is functional with coumarin (polarity sensitive motif) and morpholine , and CMP was used to real-time monitoring the polarity changes of lysosome . As for polarity A, and thBio-membranes are the basis for various compartments in live cells, so visualizing their lipid organization is helpful for studying cell status and activity . PolaritRecently, various polarity-sensitive probes for visualizing cell membrane have been exploited. Xiao et al. have developed polarity-sensitive probes (Mem-C1C18 and Mem-C18C18) with membrane-specific properties for visualizing ferroptosis . Mem-C1CAs an organelle with a large membrane structure, the endoplasmic reticulum is of vital importance to cells, and the polarity of endoplasmic reticulum needs to be visualized. Due to the urgent need for visualizing the changes of endoplasmic reticulum polarity, various probes were exploited. Smith et al. have developed an endoplasmic reticulum (ER) targeting probe through two coumarin moiety . Tang etTemperature is a crucial index in many pathological states and physiological activities , and theMitochondrial temperature plays a vital role in cells and tissues, and recent studies suggest that cancer cells have relatively hot mitochondria, which indicates that the active respire of mitochondria in cancer cells ,113. ChaThe lysosomal temperature would be changing due to the function of digesting substance. Kuang et al. have developed a lysosomal targeting fluorescent thermometer with BODIPY structure, and it was used to visualize lysosomes . The proThe lipid composition of cells (cell membrane etc.) would be adjusted when the ambient temperature changes, so monitoring cell membrane temperature changes is of vital importance to study lipid composition ,118. In Endoplasmic reticulum (ER) temperature is one of the most important parameters in ER, so visualizing the ER temperature is significant. Chang group have reported an endoplasmic reticulum probe with temperature-sensitive properties for visualizing ER temperature changes in HeLa cells . The ER The fluorescence probes for visualizing microenvironment changes in living cells and tissues could provide a useful method for biology and pathology, and it would promote the development of biology. In this review, we summarize recent discoveries in the development of microenvironment-sensitive probes. Some representative examples were displayed and classified to their sensitive microenvironment categories. We highlighted the basic design strategies, fluorescence spectra and the biological applications of these microenvironment-sensitive probes.Though great progress has been achieved for microenvironment-sensitive probes, there are also some probes need to be exploited in this area. For example, the reversible ratiometric organic probes still need to be exploited, though the “off−on” probes could solve some problems. In contrast to the organic fluorescent probes for monitoring micro-environments in an irreversible “off−on” mode, the reversible ratiometric organic probes are more convenient in imaging applications. Ratiometric organic probes enable real-time and in-situ visualization of the changes of micro-environments in dual colors, and this kind of probe could be applied for background-free bio-imaging and quantitative analysis. Moreover, the errors of instrumental fluctuations and staining operation could be eliminated by the ratiometric mode. So, reversible rationmetric organic probes will become the research focus, and more probes in this category would be exploited for visualizing and monitoring micro-environment in living cells and tissues in the near future. In addition, the single probe for dual-color monitoring two specific mico-environments and the single probe for ratiometric monitoring the same mico-environment in two organelles are still rare. Therefore, these probes would be exploited for the visualization of mico-environment in organelles, and they would prompt the development of biology in the future.In summary, fluorescence probes for visualizing microenvironments have been a powerful tool for the research of organelles, and more probes would be exploited in the near future. We anticipated this review would promote the research of organelle-related disease and denote to motivate the cancer treatment and biological areas (apoptosis or autophagy).