Controversy persists regarding many aspects of traumatic diaphragmatic hernia (TDH). We aimed to understand why some traumatic diaphragmatic injuries present with chronic hernia and to evaluate diagnosis and treatment options. Fifty acute and 19 chronic TDH patients were diagnosed and treated at our institution over a 10-year period. Clinical data from these two groups were analyzed statistically and compared. Chronic TDH patients had a significantly lower Injury Severity Score than acute TDH patients . The most common surgical approach for acute and chronic TDH was thoracotomy and laparotomy, respectively. The length of the diaphragmatic rupture was significantly shorter in chronic TDH patients than acute TDH patients . The mean length of hospital stay was significantly longer for acute TDH patients than chronic TDH patients . In conclusion, milder trauma and a smaller diaphragmatic rupture were associated with delayed diagnosis. A thoraco-abdominal computed tomography scan is needed for patients with periphrenic injuries to avoid delayed diagnosis of TDH. Improved awareness and understanding of diaphragmatic injuries will increase the rate of early diagnosis and improve prognosis. Traumatic diaphragmatic hernia (TDH) is an uncommon disease, encountered in 0.8% to 6% of blunt trauma and more than 17% of thoraco-abdominal-penetrating trauma., 7, 8 Inet al. noted that patients can experience an asymptomatic period, referred to as the “latent phase,” for decades before finally manifesting symptoms, sometimes with severe complications.[Previous studies indicate that 2.7% to 50% of diaphragmatic ruptures may not be diagnosed initially., 11 Imprications.The natural history, causes, ways to prevent delays in diagnosis, and operative approaches of TDH remain controversial. The absence of comparisons between acute and chronic TDH limits understanding of this clinical entity. Here we report our experience diagnosing and treating TDH and compare acute and chronic cases, hoping to better understand the pattern of these two clinical complaints.This study was conducted in November 2018. All 69 TDH cases were diagnosed and treated between May 2007 and December 2017 at a single institution. This study was approved by the ethics committee of the first affiliated hospital of zhejiang university. The approval number was 2018–823. According to our ethics committee, form of consent was not needed for retrospective studies. Nineteen of the cases were chronic TDH. All patients had surgical confirmation except for two chronic TDH patients who refused surgery and were diagnosed by spiral CT. We collected clinical data, including demographic information, mechanisms and severity of the injury, clinical presentation, imaging methods, characteristics of the hernia , operation approaches, length of hospital stay (LOS), and outcomes.The Abbreviated Injury Scale (AIS) is used to assess the severity of injuries in different parts of the human body, and the Injury Severity Score (ISS) is calculated based on the AIS. The ISS The definition of chronic TDH is controversial. Some authors use a specific time, such as 7 days or 1 month, as a cutoff for acute TDH, regardless of the time of the surgery or hospitalization. We definWe had no access to information that could identify any participants during or after data collection. We performed intergroup analyses using the chi-square test or Student’s t test, and P < 0.05 was considered statistically significant. We performed all analyses and calculations using SPSS .Of the 50 patients with acute TDH, 41 were male and nine were female (4.6:1). The mean patient age was 51 ± 14 years (13–77 years). A traffic accident (34 cases) was the most prevalent trauma mechanism, followed by an accidental fall (eight cases), penetrating injury (five cases), and bruising injury caused by a heavy object (three cases). Thirty-six patients had a left-sided hernia and 14 had a right-sided hernia (2.6:1). The mean ISS was 27 ± 5 (13–36). Diagnoses were made by CR, CT, and surgical exploration in 16, 28, and 6 patients, respectively. CT had a sensitivity of 93% and specificity of 100%. No patients with concern for diaphragm injury were confirmed negative by surgical exploration.Of the 19 patients with chronic TDH, eight were male and 11 were female (0.7:1). The mean patient age was 52 ± 17 years (32–85 years). At the initial injury, 13 patients were asymptomatic, and six experienced slight to moderate abdominal or chest pain. In the chronic phase, 14 patients complained of abdominal discomfort, including abdominal pain (nine cases), nausea and/or vomiting (four cases), and hematemesis (one case). Two patients complained of chest tightness and chest pain. Three patients were asymptomatic. The median duration of symptoms was 7 days (1–900 days). The most prevalent trauma mechanism was a traffic accident (13 cases), followed by penetrating injury (four cases) and an accidental fall (two cases). The interval between the trauma and diagnosis was 8 ± 7 years (1–30 years). Fourteen patients had a left-sided hernia and five had a right-sided hernia (2.8:1). The mean ISS of the previous injury was 10 ± 3 (5–17). The diagnosis was confirmed by CT in all patients. The sensitivity and specificity were both 100%All of the acute TDH patients underwent surgery to ameliorate herniated organs and repair the diaphragm by non-absorbable suture. The operative approaches were posterior thoracotomy in 26 patients, combined laparotomy and midline thoracotomy in eight patients, midline laparotomy in six patients, left subcostal laparotomy in six patients, combined posterior thoracotomy and left subcostal laparotomy in two patients, and midaxillary line thoracotomy in two patients. The mean length of the diaphragm rupture was 11 ± 3 cm (4–20 cm). The most common herniated organ was the stomach (22 cases), followed by the omentum (20 cases), liver (14 cases), colon (nine cases), spleen (eight cases), small intestine (six cases), and cholecyst (three cases).Two chronic TDH patients rejected surgical treatment. One was asymptomatic and the other felt slightly oversatiated after meals. Seventeen patients underwent surgery to repair the diaphragm by non-absorbable suture. The operative approaches were midline laparotomy in 10 patients and posterior thoracotomy in seven patients. An additional left subcostal laparotomy was added to the posterior thoracotomy for one patient because the incarcerated and necrotic intestines needed to be excised. Two patients who underwent midline laparotomy were given additional surgery besides diaphragm repair. One patient had an additional incision decompression of the intestine, and another patient had a colostomy after resection of the left colic flexure. The mean length of the diaphragm rupture was 6 ± 2 cm (4–11 cm). The most common herniated organ was the colon (nine cases), followed by the omentum (seven cases), stomach (five cases), small intestines (three cases), liver (three cases), spleen (three cases), and cholecyst (one case).The mean LOS was 41 ± 31 days (5–198 days) for acute TDH patients. Postoperative complications included pneumonia or atelectasis (n = 6), urinary tract infection (n = 4), septicopyemia (n = 3), infection of surgical incision (n = 2), renal failure (n = 2), ileus (n = 1), and pulmonary thromboembolism (n = 1) The death rates for left-sided and right-sided TDH patients were 8.3% (n = 3) and 14.3% (n = 2), respectively. Reasons for death were septicopyemia (n = 2), multiple organ failure (n = 1), respiratory failure due to severe pneumonia (n = 1), and hematencephalon or cerebral infarction (n = 1). The mean follow-up time was 5 ± 4 years. At the end of follow-up, only one patient had a recurrence and underwent another posterior thoracotomy to repair the re-ruptured diaphragm. Mesh was used to reduce tension.The mean LOS was 17 ± 10 days (5–35 days) for the chronic TDH patients. The three patients who had additional surgeries other than diaphragm repair had the longest hospital stays . The mean follow-up time was 6 ± 4 years, and no patient had a recurrence at the final follow-up date.There was a significant difference in the male/female ratio between acute and chronic TDH patients. Males accounted for 82.0% of acute TDH patients but only 42.1% of chronic patients. Acute TDH patients were mostly admitted by the emergency department, whereas chronic TDH patients were mostly admitted by the outpatient service . The length of the diaphragm rupture was significantly shorter in chronic TDH patients than acute TDH patients . The mean LOS was significantly longer for acute TDH patients than for chronic patients .TDH remains challenging for clinicians. The main cause of TDH largely depends on geographic and socioeconomic factors., 8 At ouIn our experience, respiratory symptoms are more prevalent for patients with acute TDH. However, it is difficult to study clinical symptoms in the acute phase, as patients may be unconscious or the symptoms may be subtle. For chronic TDH patients, gastrointestinal complaints caused by volvulus or incarceration of the intra-abdominal viscera are more common than respiratory symptoms (7:1). We also found that there was a higher rate of intestinal hernia in the chronic phase than in the acute phase. This may be attributable to the anatomical structures of the intra-abdominal organs and peristalsis of the intestines. Therefore, when patients have abdominal complaints, clinicians should refer to both recent and past trauma histories. Chronic TDH should be diagnosed promptly, because obstruction and strangulation of the intra-abdominal organs increase morbidity and mortality.et al. proposed that the rupture is enlarged during the latent phase,[Our study shows that diaphragmatic ruptures of chronic TDH are smaller than those of acute TDH. Grimes nt phase, and thusIt was previously thought that, in emergency situations, the symptoms of diaphragmatic hernia are obscured by other more dominant or life-threatening comorbidities. However, our results contradict this assumption. Patients with milder trauma are less likely to have a large rupture with hernia, leading to no symptoms or radiological abnormalities.Another suggestion is that, for right-sided diaphragmatic ruptures, the liver may not herniate into the thorax in the acute phase but may continue to serve a protective role that limits protrusion of intra-abdominal organs. In our series, all cases of acute, right-sided TDH presented with liver hernia, whereas only half of the chronic TDH patients had liver hernia. This is consistent with the above theory., 21, 22et al. showed that diaphragmatic injuries cannot be excluded until extubation is achieved.[Because of a variety of diagnostic pitfalls, diaphragmatic rupture is often overlooked in patients with thoracoabdominal injury. In our sachieved. We also Although some diaphragmatic ruptures may be very difficult to diagnose initially, close cooperation between clinicians and radiologists may prevent missed diagnoses. In our study, CR was diagnostic in 32% of acute cases, which allowed for timely surgery. CT was the most common diagnostic strategy in both acute and chronic TDH patients. Previous studies indicate that admission supine CR is the most commonly performed diagnostic study after trauma and establishes a correct diagnosis in 27% to 62% blunt-trauma TDH patients. In 18% of cases, CR is suggestive but not diagnostic because of the small wound size of penetrating trauma, superimposed pathology around the diaphragm, and the protective function of the liver. Many of Despite the differences in clinical presentation between acute and chronic TDH, a careful physical examination, diagnostic imaging study, and even endoscopic exploration , 4 are iOnce the a diagnosis of TDH is made, surgery to reduce the herniated organs and repair the diaphragm is mandatory. If the pA limitation of this study is its small sample size and retrospective study design, which made it difficult to control the homogeneity between different groups. The small number of penetrating cases may have led to type II error. Patients who died before being admitted to the hospital were not included in this study, which may have introduced selection bias. Meanwhile, given the long time interval between the initial injury and the manifestation of TDH, some first-visit clinical data were not acquired.Milder trauma is more likely to result in chronic TDH, a finding that contradicts the published literature. Thoraco-abdominal CT scan should not be excluded for patients with periphrenic injuries and low ISS. Awareness and understanding of diaphragmatic injuries should be improved in trauma centers to avoid delayed diagnosis.S1 Data(SAV)Click here for additional data file.


The treatment of adult refractory idiopathic membranous nephropathy with steroid and other immunosuppressant-resistant nephrotic syndromes can be a significant challenge. We evaluated the efficacy and safety of the traditional Chinese medicine Jian Pi Qu Shi Formula (JPQSF) as a promising regimen.We analyzed 15 consecutive patients with biopsy-proven idiopathic membranous nephropathy who failed immunosuppressive therapy from October 2013 to January 2017. JPQSF was administered orally two times per day, respectively, in the morning and at night for 6 months. All patients had at least 1 year of follow-up. The primary endpoints included complete or partial remission. Secondary endpoints included change of clinical parameters and adverse events after 12 months of treatment. After 12 months, complete remission was achieved in 13.3% of patients and partial remission in 66.7%, yielding a response rate of 80%. Proteinuria, serum albumin, and cholesterol were improved significantly . After 1 year of treatment, proteinuria (mean ± SD) decreased from 5.93 ± 2.54 g per 24 h to 1.99 ± 1.17 g per 24 h (P<0.001). No serious adverse events occurred during the observation.resistant membranous nephrotic syndrome patients, with a favorable safety profile. Larger and longer follow-up studies evaluating this regimen are warranted. JPQSF may be an alternative therapeutic option for steroid and general immunosuppressant- Idiopathic membranous nephropathy (iMN), the major cause of nephrotic syndrome in Chinese adults , has dra zang-fu organs as its basis and differentiation of syndromes and treatment as its diagnostic and therapeutic features. The practice of traditional medicine is based on the theory of TCM whose basic premise is the idea of harmony. Through cumulative empirical experience of experts, patients with iMN have been treated with different formulations of herbs such as Astragalus membranaceus and Angelica sinensis, demonstrated to be effective on proteinuria in animal studies [Traditional Chinese medicine (TCM) practitioners have been treating kidney diseases for several centuries in China and other countries in Asia and have accumulated extensive clinical experiences , 9. TCM  studies , 11. She studies , but theJian Pi Qu Shi Formula (JPQSF) has been developed for the treatment of iMN in Xiyuan Hospital, affiliated with China Academy of Chinese Medical Science. There are 10 herbs in JPQSF, which are selected based on the theory of traditional Chinese medicine and extensive experience from Professor Ren-huan Yu who has been practicing traditional Chinese medicine to treat patients with kidney disease over several decades. JPQSF is used to invigorate spleen and dispel dampness, thus restoring the harmony of Yin and Yang.We here present a retrospective analysis of the outcomes of 15 consecutive RMN patients treated with JPQSF to examine the therapeutic efficacy of this regimen.This was a retrospective case-series study, including all patients with histologically proven membranous nephropathy presenting to the Department of Nephropathy in Xiyuan Hospital.⩾3.5 g per 1.73 m2 per 24 h, a serum albumin<3 g per L, and peripheral edema and are on optimal therapy with angiotensin converting enzyme inhibitors and/or angiotensin receptor blockers. Patients were excluded if they had any of the following conditions: (1) other types of membranous nephropathy, such as secondary membranous nephropathy, which were ruled out by obtaining patients' medical histories of malignancy, review of medications for potential drug use, contact with poison and by screening for laboratory tests, such as for hepatitis B and C viruses and antinuclear antibody testing; (2) diabetes or hemoglobin A1c level >6.2 mmol per L; (3) those who had received steroids or immunosuppressive drugs but with lack of standardization; (4) other types of syndromes in patients treated at Xiyuan Hospital, such as kidney Qi deficiency, Qi and Yin deficiency, stagnation of phlegm, and blood stasis. The present study was approved by the Ethics Committees of Xiyuan Hospital. In view of the retrospective nature of the study, a collection of informed consent was abandoned.This study was conducted on patients with histologically proven membranous nephropathy presenting to the Department of Nephropathy in Xiyuan Hospital between October 2013 and January 2017; they were screened and selected based on following criteria: (1) already failed a regimen of cyclical monthly steroid and cyclophosphamide and/or CNI to induce a partial remission after at least six months of therapy; notably, all the patients have stopped such treatment before the study; (2) according to the syndrome of spleen deficiency and dampness excess; (3) medical records materials being completed. In accordance with “the guiding principles for the clinical study of new drugs for use in traditional Chinese medicine” released in 2002 and TCM theory of combination of disease and syndrome, combined with years of clinical observation and experience in our department, we summarized the standards of spleen deficiency and dampness excess syndrome in RMN, being formulated as follows: Primary symptoms are mental fatigue, soreness of waist, and edema. Secondary symptoms are (1) reduced appetite, insufficiency with chills, loose stools, or diarrhea and (2) dim complexion, frequency of urination, or frequency of urination at night. Tongue and pulse symptoms include (1) enlarged and tooth-marked tongue and greasy coating and (2) slow and sunken pulse. Participants can be diagnosed with spleen deficiency and dampness excess syndrome if they meet primary symptom and one of secondary symptoms and tongue or pulse. Patients were considered to have the nephrotic syndrome if they had a proteinuriaEligibility required a supportive treatment that was adjusted to ensure that they were receiving the maximum labeled or tolerated dose of RAS blockade, along with optimized blood pressure control according to 2012 KDIGO guidelines for glomerulonephritis and hypertension. Patients with syndrome of spleen deficiency and dampness excess were prescribed JPQSF. JPQSF is made of 10 different Chinese herbs . It was All patients were followed up at regular intervals at least one year. Patients were scheduled to have study visits at baseline and months 3, 6 and 12. Additional telephone or face-to-face (at the choice of the investigator) visits were required if patients were not available. At each study visit, clinical and laboratory parameters including 24 hour urinary protein, serum albumin, plasma creatinine, liver enzymes , and blood cell counts were measured. ⩾50% reduction compared with baseline. Any patient reaching a CR or PR was considered a treatment success.The primary outcome was attainment of remission rate, including complete and partial remissions. Remission was defined according to 2012 KDIGO guidelines as follows: (1) complete remission (CR) was defined as a reduction in proteinuria to protein excretion 0.3 g per 24 h. (2) Partial remission (PR) was defined as the case of urinary protein excretion < 3.5 g per 24 h withThe secondary outcomes included the change in proteinuria, serum albumin level, evaluated glomerular filtration rate (eGFR), doubling of serum creatinine level, end-stage renal disease, and death. Because albumin level may be an earlier marker of response than end points defined by proteinuria only , we alsoAny symptoms and adverse effects such as infections, hospitalizations, or other complications were recorded at each follow-up visit or immediately when this happened. Serious adverse events (SAEs) were defined according to the International Conference on Harmonization Guideline for Clinical Safety Data Management.Baseline characteristics of the study population were presented as frequencies and percentages for qualitative variables and medians and interquartile ranges (IQR) for continuous variables. Remission rates were expressed as frequencies or percentages and their 95% confidence intervals (95% CIs). For the comparison between two groups among the three groups, Bonferroni's correction of the Mann-Whitney test was used. Statistical analyses were carried out using SPSS, version 22. All P values were 2-sided, and P values less than 0.05 were considered statistically significant.From October 2013 to January 2017, 15 consecutive patients with RMN receiving JPQSF were enrolled. All the patients received maximal tolerable doses of angiotensin receptor blockers  and atorvastatin.There were 5 female and 10 male patients, with an average age of 47.6 ± 8.4 years. All the subjects had been previously administered and failed at least six-month course of immunosuppressant treatment: 6 patients resistant to corticosteroid plus cyclophosphamide, cyclosporine in 7 patients with relapsing disease, and 2 patients intolerant to tacrolimus treatment .2 . Serum albumin was 23.88 ± 3.28 g per L. Serum creatinine was 80.33 ± 26.42 umol per L and the mean creatinine clearance was 96.48 ± 28.91 ml per min per 1.73 m2 . All the2 .In the 15 patients, being treated with JPQSF for 6 months, the remission rate was 66.7%; the CR was achieved in one patient and PR in nine patients , and pro2), renal function was improved and stable (68.84 and 42.17 ml per min per 1.73 m2).The reduction in proteinuria was paralleled by a progressive and significant increase in serum albumin levels from 23.88 ± 3.28 g per L at baseline to 38.96 ± 9.08 g per L at 12 months  has been used as an effective agent for iMN with SRNS , 15 and However, these therapies do not always induce remission and also cause significant adverse effects; regimen like rituximab or tacrolimus may modulate immunity and increase susceptibility to infection like Pneumocystis jiroveci pneumonia  and otheTCM practitioners have been treating kidney diseases for several centuries in China and other countries in Asia and have accumulated extensive clinical experiences . The oveWith recent integration of TCM and western medicine, diagnosis of CKD by practitioners of TCM has supplemented traditional diagnostic approach with western molecular and imaging diagnostic tools. The current treatment of CKD in TCM is often achieved by combining TCM and western pharmacologic agents. The therapeutic principles of TCM for CKD include ‘replenishing vital energy and nourishing blood,' ‘clearing heat and eliminating dampness,' and ‘coordinating Yin and Yang in the body' . Hundred Abelmoschus manihot (L) medic (AM) [ Tripterygium wilfordii Hook F (TWHF), triptolide. In an uncontrolled trial, Chen et al. [ triptolide in children with moderately severe Henoch-Schönlein purpura concluded that triptolide is effective in relieving short-term symptoms.Although many small clinical studies published in Chinese journals support a kidney protective effect of traditional medicine for CKD, large and well-designed randomized controlled trials (RCTs) are still lacking. However, progress has been made recently in this field. Large randomized clinical trials of TCM in kidney disease include Shenqi particle and Huangkui capsule. To our knowledge, Shenqi particle  was the dic (AM)  in patien et al.  examined Tripterygium preparations, TWHF, Astragalus, Rheum officinale (a type of rhubarb), standard care, or other immunosuppressive treatment in patients with CKD. On account of the small sample size, short follow-up, and concerns for methodologic bias, the quality of evidence was suboptimal, and there is insufficient evidence to conclude that such TCM regimens are as effective as other regimens. Therefore, large RCTs are required to confirm these findings.Recently, several systematic reviews and meta-analyses were performed on RCTs using different Astragalus membranaceus, TWHF, Saikosaponin A, radix puerariae (kudzu root), AM, and Cortex moutan (root bark of Paeonia suffruticosa) both in vitro and in vivo trials. Some traditional medicines like Astragalus, TWHF, Saikosaponins, and Rhein (an acidic compound derived from rhubarb) have also been reported to have antifibrotic effects and to inhibit mesangial cell proliferation and matrix synthesis. In addition, several traditional medicines such as Astragalus, Triptolide, Saikosaponins, and Emodin are known to regulate the immune system [Mechanisms of action have been studied for some herbs. Their effects are mainly related to anti-inflammation, antioxidation, antifibrosis, regulation of immune system, anticoagulation, and improvement of metabolic disturbance . Active e system . TherefoOwing to the rapid economic growth and scientific development over the past decade, the Chinese government has supported studies to examine the scientific basis of TCM using advanced cell and molecular biology approaches. A rapid development of TCM is expected over the next decade. Nowadays more and more animal studies have confirmed the biological activity and therapeutic effects of several traditional Chinese herb medicines in CKD.In the present study, the traditional Chinese medicine formulation of JPQS decoction was effective at inducing remission in patients with RMN. In addition, JPQSF had a safety profile. Astragalus. Astragalus has been shown to have strong anti-inflammatory and antioxidative stress effects in different cell types, including kidney cells, through suppression of p38 MAPK (mitogen-activated protein kinase), NF-κB (nuclear factor κB), and Toll-like receptor–mediated pathways [ Astragalus has been shown to reduce proteinuria and attenuate kidney injury in several animal models of kidney disease [ Astragalus extract in patients with different forms of chronic glomerulonephritis has been shown to reduce proteinuria [ Dioscoreae Nipponicae Rhizoma has shown potent anti-inflammatory and antiarthritic effects in vitro and in vivo studies [κB expression. The role of other herbs in renal protection requires further study.JPQSF contains 10 different herbs and the exact active components for the antiproteinuric effect are unclear. However, the therapeutic activity of JPQSF could be related to the herbal componentpathways , 29. Astteinuria , 18 and teinuria . Dioscor studies . It has In the present study, cases with severe adverse events were not observed. The safety profile of JPQSF suggests an important advantage of using TCM in RMN patients and/or elderly patients with other severe illness because these patients are more susceptible to the adverse effects of immunosuppressive agent. Astragalus and Angelica, two components of JPQSF, have been shown to improve protein metabolic disorder in patients with nephrotic syndrome by promoting net protein synthesis. These studies suggest that JPQSF may improve serum albumin levels through these mechanisms.One of the findings in this study was that a significant increase in serum albumin level before improvement in proteinuria was observed, which suggests that the mechanism underlying JPQSF's initial effect on plasma albumin level might be independent of the reduced urinary protein excretion.Our study has several important limitations. The major limitation of this study is that we were not able to follow up these patients for a longer period to observe the complete remission rate and relapsing rate because these patients are from different areas of China. Another limitation is that the exact active components of JPQSF and mechanisms of action remain unclear. This study is retrospective single-center, of limited size, and lacks a comparator group. Statistical results must be interpreted with caution in this setting. Larger studies are required to confirm our findings.In summary, JPQSF was effective and safe for the treatment of RMN and may be an alternative therapeutic option for the treatment of refractory nephrotic iMN patients who failed to respond to steroid and other conventional immunosuppressant agents. Over a follow-up time of 1 year, 80% of patients achieved remission. Future randomized clinical trials with long-term follow-up are inevitably needed to establish JPQSF as evidence-based treatment option in this part of refractory nephrotic syndrome.


Organic and inorganic xenobiotic compounds can affect the potential ecological function of the soil, altering its biodiversity. Therefore, the response of microbial communities to environmental pollution is a critical issue in soil ecology. Here, a high-throughput sequencing approach was used to investigate the indigenous bacterial and fungal community structure as well as the impact of pollutants on their diversity and richness in contaminated and noncontaminated soils of a National Interest Priority Site of Campania Region  called “Terra dei Fuochi”. The microbial populations shifted in the polluted soils via their mechanism of adaptation to contamination, establishing a new balance among prokaryotic and eukaryotic populations. Statistical analyses showed that the indigenous microbial communities were most strongly affected by contamination rather than by site of origin. Overabundant taxa and Actinobacteria were identified as sensitive biomarkers for assessing soil pollution and could provide general information on the health of the environment. This study has important implications for microbial ecology in contaminated environments, increasing our knowledge of the capacity of natural ecosystems to develop microbiota adapted to polluted soil in sites with high agricultural potential and providing a possible approach for modeling pollution indicators for bioremediation purposes. In fact, soil pollution could cause pressure on sensitive microorganisms and thus could change the composition of microbial community4. Although organic pollutants have been shown to reduce microbial biodiversity5, they can be used as a carbon source by some species of microorganisms6, thereby stimulating their growth in contaminated soil and thus leading to the development of a new microbial community diversity7. However, little is known about the microbial response to multicontamination and remediation practices due to the high biodiversity of microflora as well as to the complex relationships among microbial communities and biotic and abiotic processes influencing their activities in soil8. Therefore, investigating and understanding the interactions between microorganisms and soil components will assist us in exploring and establishing the potential relevance between soil microorganisms and microbial processes9. The use of novel approaches based on culture-independent high-throughput sequencing can reveal uncultivable microbiota and enables the study of microbial ecology and taxonomic diversity at a high resolution10, allowing a broader range of comparisons between different soils with varying levels of contaminants. Moreover, this approach could also be useful for identifying specific microbial biomarkers that could be used as indicators for the ecological status and health of soils as well as for possible biotechnological applications for bioremediation plans. Various studies have been conducted to investigate the impact of contamination on the indigenous microbial populations and their shift in order to discover phylogenetic markers with potential degradative abilities. These studies revealed different microbial populations organized in complex communities on the basis of the environmental conditions. In particular, contrasting observations of the impact of contaminants on microbial diversity due to many factors that are involved in the microbial response to pollutants have been reported11. Proteobacterial populations were dominant and recurrent in petroleum- and polycyclic aromatic hydrocarbons (PAHs) -polluted soils13 and in coastal sediments14 as well as in uranium mines16. Fragoso dos Santos et al.17 indicated possible targets for the biomonitoring of the impact of oil in mangroves. The order Chromatiales and the genus Haliea were detected as sensitive indicators, while the three genera Marinobacterium, Marinobacter and Cycloclasticus were reported as resistant taxa. Jeanbille et al.18 identified prokaryotic and eukaryotic potential biomarkers for PAH chronic contamination in coastal sediments. Additionally, the history of pollution plays a crucial role in the microbial community structure. In fact, no general trend has emerged yet, and the short-term impact of contamination tends to decrease microbial abundance, richness and diversity, while in aged or chronically contaminated environments, a surprisingly high bacterial diversity, due to adaptation over time and stability caused by long-term exposure, has been observed18.Soil contamination generally affects the potential ecological function of the environment, altering soil functioning, health and biodiversity19 and are actually identified by the Italian State as Regional Interest Priority Sites (RIPS)20.In this context, the aim of this study was to determine and describe the native microbiota and the impact of anthropogenic pollution  on the diversity and richness of prokaryotic and eukaryotic communities occurring in soils of two rural sites of Campania  subject to illicit waste disposal and dumping or suspected to be polluted by metals due to agricultural practices. Both these sites are located in an area formerly classified as National Interest Priority Sites (NIPS)The studied soils were characterized by very similar chemical properties and particle size distribution.2O was neutral in G (pH 7.3 ± 0.2) and subalkaline in TD (pH 8.0 ± 0.2), in agreement with the carbonate content (8 ± 2.5 g kg−1 in G vs. 174 ± 41 g kg−1 in TD). The electrical conductivity was always below the values that limit plant growth and agricultural production . The soil organic carbon content was never above 2%, with similar values in both sites . The cation exchange capacity (CEC) was 28 ± 3.1 cmol(+) kg−1 in TD and 34 ± 3.2 cmol(+) kg−1 in G, with a dominance (~80%) of calcium in the exchange complex. Therefore, the physical-chemical fertility of both the soils did not result in limiting plant growth and did not show any features that may alter the microbial conditions.Both soils were sandy loam, with 61%  and 71%  of the soil in the sand fraction. Soil pH-H−1, respectively. Likewise, the concentration of heavy hydrocarbons (C > 12) in both G and TD soils was well above the Italian threshold of 50 mg kg−1, while for PAHs, only benzo(a)pyrene was found, in few cases, at concentrations equal to or slightly above the threshold of 0.10 mg kg−1.It was found that the soils of four on seven plots for both G and TD sites can be considered potentially contaminated for residential use, in accordance with Italian environmental law (Law Decree 152/2006) , Cu  and Zn , while the remaining 6 plots (3 in G and 3 in TD) were considered not contaminated.Table The microbial diversity from G and TD was characterized by partial 16S and 18S rRNA gene sequencing obtained from DNA directly extracted from soil samples of noncontaminated (NoCont) and long-term contaminated (Cont) plots. In total, 4,613,050 and 1,226,128 high quality reads were analyzed for prokaryotes and eukaryotes, respectively. The alpha-diversity was determined by calculating the Shannon diversity index and the Chao1 richness index based on OTUs of 97% identity.vs. GNoCont and TDCont vs. TDNoCont, P < 0.001). The presence of environmental stressors such as heavy metals strongly reduced the total bioactivity, richness and diversity of microorganisms with increasing pollutant concentrations in the soil22.As shown in Fig. vs. TDCont, P < 0.001) indexes  with the Shannon index  and richness (P < 0.05) when comparing GCont and TDCont samples . This difference increased when the two factors were combined. In fact, ANOSIM showed a significant difference for site-origin x contamination (P < 0.001), demonstrating a correlation between the contamination and the site of origin of samples.As shown in Fig. Relative abundances of bacterial and fungal taxa were examined at the phyla and class level to determine whether there were any significant shifts in the composition of the microbial communities according to the site-origin and contaminated samples.et al.25 reported that these taxa were among the dominant phyla that significantly increased in petroleum hydrocarbon-contaminated soils and proved to be the most influential on the biodegradation of these pollutants. Moreover, consistent with previous studies, a shift to Proteobacteria dominance  in hydrocarbon26 and heavy metal27 polluted soils was observed.In total, forty-six different bacterial phyla were detected in the soil samples, but only Actinobacteria, Proteobacteria, Acidobacteria, Firmicutes, Bacteroidetes, Gemmatimonadetes, Planctomycetes, Chloroflexi and Verrucomicrobia were detected with an incidence >1% in at least one sample, accounting for approximately 93–97% of the total biodiversity in each sample Fig. . AlthougBy contrast, the phyla Actinobacteria, Firmicutes, Gemmatimonadetes and Chloroflexi showed an opposite trend, strongly decreasing in contaminated soils  with respect to uncontaminated soil, suggesting that they have low adaptability to contamination. Yin et al.34 reported that Actinobacteria were susceptible to heavy metals, and other studies showed that various bacterial populations, especially actinomycetes, were negatively correlated with metals, which drastically reduced their growth36.Although these taxa, especially Actinobacteria, are known to have specific hydrolytic enzymes for the decomposition of a wide variety of organic materials37 as well as vanadium38. Other eukaryotic phyla, such as Basidiomycota, Oomycota, Chytridiomycota and Mucoromycota, were found to a lesser extent. Their abundance varied as a function of site of origin (G or TD), and within each site, abundance was influenced by the presence of contaminants, except for Mucoromycota, which remained quite stable  than in noncontaminated soils (approximately 1%) Fig. . Members44. Among the other eukaryotic classes recovered to a lesser extent, Eurotiomycetes and Chytridiomycetes decreased approximately 2-fold in contaminated soils, dropping to 6% and 4% and 1.5 and 2% in GCont and TDCont samples, respectively, compared to unpolluted soils . Dothideomycetes showed a similar trend, although a marked decrease was observed only in TDCont (2%) compared to TDNoCont (9%). Conversely, the abundance of Peronosporomycetes strongly increased in GCont (12%) compared to unpolluted GNoCont (5%), whereas its percentage remained quite stable in TD  in the abundance for different taxonomic rankings, allowed the identification of characteristic biomarkers of contaminated and noncontaminated soils at the sites TD and G. For the site G, the cladogram revealed 313 differential bacterial OTUs, of which 162 and 151 were detected in polluted and unpolluted soil, respectively  were represented by Alphaproteobacteria, Betaproteobacteria, Planctomycea and Sphingobacteria. Proteobacterial communities, mainly composed of Alpha-, Beta- and Gammaproteobacteria, might be involved in the biodegradation or biotransformation of numerous organic compounds, although their proportions varied among different polluted environments46. An interesting finding was that all OTUs classified as Betaproteobacteria in polluted soils were identified as methylotrophic bacteria, which are known to have great potential in the bioremediation of environmental pollutants such as chlorinated solvents and methyl tert-butyl ether (MTBE)47 as well as PAHs48.Since PCoA analysis on weighted UniFrac matrixes highlighted a strong phylogenetic diversity of the microbiota in contaminated soils influenced by the site-origin of samples, to identify potential bacterial and fungal biomarkers regardless of the site-origin of contaminated soil samples, Venn diagram analysis was carried out among biomarkers. As shown in Fig. 7 and to rank among the top taxa in oil-contaminated soil49. In addition, OTUs identified as Sphingobacteria and Acidobacteria were other bacterial biomarkers recovered in polluted soils. These taxa were previously recovered from plant biomass-degrading microorganisms ecologically related to the soil ecosystem52, highlighting their ability to synthetize a wide number of enzymes for the depolymerization of recalcitrant organic matter. Moreover, unculturable bacteria, such as candidate division agg27, CH21, TM7_3 and ZB2, emerged as new biomarkers of polluted soils. In fact, among taxa that drive the biodegradation of hydrocarbons in natural soils, uncultured bacteria could play a key role8. The other bacterial biomarkers of polluted soils were identified as Anaerolineae, Bacilli, Chlamydiae, Opitutae, Phycisphaerae, Spartobacteria and Verrucomicrobiae. Sutton et al.5 reported that Anaerolineae was associated with anaerobic degradation of oil-related compounds and that its presence in soils could be related to the natural attenuation under anoxic conditions. Verrucomicrobia was recognized as the most abundant phylum in an oil-contaminated soil sampled from major oilfields in Northern China49, and it was ubiquitous in multiple petroleum-contaminated soils53. Finally, among eukaryotes, Basidiomycota, particularly Malasseziales, could be considered a fungal biomarker in contaminated soils. Although species belonging to this taxon remain uncultured, they were previously recovered at the later stages of the composting process of recalcitrant materials54 as well as from deep-sea extreme environments57.Members of the phylum Planctomycetes were reported to be highly correlated with high copper and lead concentrations in contaminated soils61, the results obtained in this work showed that they are sensitive to stress conditions due to the presence of high multicontamination in a complex natural environment, such as soil. Dias et al.62 reported that a large amount of fresh diesel in a contaminated soil led to unfavorable environmental conditions for the growth of Actinobacteria, which increased only at the final stage of the bioremediation process, when the levels of available hydrocarbons were low. In addition, a wide variety of species found within Actinobacteria highlighted that under unstressed conditions, no selection pressure occurred at either site.In unpolluted soils, 101 bacterial taxa belonging to 9 classes Fig.  and 8 fustatus and health of agricultural soils as well as for possible biotechnological applications were identified. In particular, Actinobacteria represents a sensitive biomarker for assessing soil pollution and therefore could provide general information on the health status of the environment. This approach could also be useful for bioremediation purposes to identify autochthonous populations for isolating microbial degraders from contaminated soils with specific xenobiotic toxic compounds. Therefore, this work improves the knowledge about the responses of indigenous microbial populations to anthropogenic activities, particularly related to the potential ecological risks of contamination in arable lands and the capacity of natural ecosystems to develop a microbiota adapted to polluted soil. Such information cannot be ignored in the evaluation of the adverse effects of potentially toxic metal distribution and accumulation trends in agricultural soil. Ecological risks, along with the more commonly assessed human health risks, could aid in strategic planning and management aimed at reducing soil contamination.In conclusion, this study has important implications for the microbial ecology of soil belonging to the Regional and National Interest Priority Sites. Even if the response of the microbial communities to environmental stresses is a critical issue in soil ecology, our results showed that microbial population composition shifted significantly in contaminated environments. The microbial populations were sensitive to pollution, since the relative abundance of different taxa strongly changed, establishing a new prokaryotic and eukaryotic community in the contaminated soils. In addition, potential biomarkers that could be used as indicators for the ecological 11 and to understand the capacity of microbial populations to colonize the soil ecosystem to recover natural biofertility. In fact, evolutionary studies would not only improve the knowledge of the microbial community ecology in contaminated environments but also allow the implementation of potential bioremediation strategies to address the increasing impacts of xenobiotics in the ecosystems63. However, further studies are needed to determine the resistance/tolerance and molecular mechanisms involved in the adaptation of spontaneous microbial biodegraders to contaminated and noncontaminated agricultural soils.Investigating the microbiota of multicontaminated agricultural soils could represent a good opportunity to clarify microbial adaptation with important applications in the field of bioremediation and/or biostimulation64. The two study sites  were located 3 km apart in a potentially contaminated plain of Campania region19 and therefore characterized by Mediterranean climatic conditions (rainy/temperate autumn-winter and arid/ warm spring-summer).The study sites were two fallow rural fields that were used in the past for illegal waste disposal and dumping: Giugliano-NA and Trentola Ducenta-CE. They were used as pilot fields in the LIFE-Ecoremed project aimed to validate eco-compatible techniques for soil remediation65. The soil samples were analyzed for heavy hydrocarbons (C > 12), several PAHs and PTEs. Details of the soil sampling scheme based on a two-level grid resolution to assess pollutant spatial distribution are described by Monaco et al.66 and Rocco et al.67. The Giugliano site was mainly polluted by heavy hydrocarbons (71% of the area), Cu  and Zn (12% of the area); the Trentola-Ducenta site was polluted by heavy hydrocarbons  and Cu (7% of the area)68. Based on these results and taking into account the spatial distribution of pollution, 14 plots (7 in Giugliano and 7 in Trentola-Ducenta) with different pollution levels were selected for microbial analysis. Form these plots, 1 kg soil samples were collected, homogenized and sent to the microbiological laboratory.After waste removal, soils were sampled on December 2013 under the following environmental conditions : 23, 10and 16 °C for maximum, minimum and mean air temperature, 80% for mean air relative humidity and a cumulative rainfall of 71 mmaqua regia followed by inductively coupled plasma-atomic emission spectrometry (EPA 3051 A and EPA 6020 A). Heavy hydrocarbons (C > 12) were determined according to UNI EN ISO 16703. For PAH determination, the US-EPA method 8270D was applied with a gas chromatograph coupled to a quadrupole mass spectrometer (GC/MS). In Italy, soil quality standards for agricultural areas have not yet been established. Therefore, all data were referenced against the threshold limits imposed by the Italian Action Levels for Residential land use (IALR) established under Italian environmental law (D.Lgs 152/2006) and the local Soil Baseline Reference values70.Soil samples were analyzed for main chemical properties  and particle size analysis. The pseudodototal content of 13 PTEs was measured using microwave-assisted acid digestion in et al.66 and Rocco et al.67.More details on the soil analysis and pollutant extraction methods are reported by Monaco Total genomic DNA was extracted using a FastDNA SPIN Kit for Soil  according to the manufacturer’s instructions.71 for the bacterial V3-V4 region of the 16S rRNA gene and the primers NS1 (5′-GTAGTCATATGCTTGTCTC-3′) and NS2 (5′-GGCTGCTGGCACCAGACTTGC-3′)72 for the fungal 5′-end of the 18S rRNA gene. Amplicon purification, multiplexing and sequencing were carried out by Genomix4 Life s.r.l.  as reported in the Illumina Metagenomic Sequencing Library Preparation manuals. Sequencing was carried out on a MiSeq platform , leading to 250 bp or 300 bp paired-end reads for bacteria and fungi, respectively.The microbial diversity was evaluated by amplicon-based metagenomic sequencing using the primers S-D-Bact-0341F50 (5′-CCTACGGGNGGCWGCAG-3′) and S-D-Bact-0785R50 (5′-GACTACHVGGGTATCTAATCC-3′)73. Low-quality reads (Phred score <20) were trimmed, and reads shorter than 60 bp were discarded in end-to-end, sensitive mode.Row reads were quality analyzed and filtered using PRINSEQ.74, and sequences were then analyzed using QIIME 1.9.1 software75. Operational taxonomic units (OTUs) at 97% sequence identity were picked through a de novo approach, and the uclust method and taxonomic assignment were obtained using the RDP classifier and the Greengenes76 or SILVA SSU/LSU77 database for 16S and 18S rRNA sequences, respectively. Chloroplast contamination was removed from 16S OTU tables, while only Fungi and Peronosporomycetes sequences were retained in 18S OTU tables. Singletons were also discarded from filtered OTU tables, and the relative abundance of other taxa was recalculated. To avoid biases due to different sequencing depths, OTU tables were rarefied at the lowest number of sequences per sample.Paired-end reads were merged using FLASHhttp://www.r-project.org). A hierarchical clustering heatmap was obtained through the made4 package using the weighted pair group method with arithmetic mean (WPGMA) method. Alpha (Chao1 richness and Shannon diversity indexes) diversity analysis was computed by QIIME on rarefied OTU tables, and the pairwise Wilcoxon test was used in R to test for significant differences. Beta diversity was calculated and plotted using the phyloseq R package78 for the Weighted UniFrac, and ANOSIM statistical analysis was then carried out with the Vegan R package79 on the same distance matrix.Statistical analyses and plotting were carried out in R  effect size (LEfSe)The raw Illumina sequencing data are available in the Sequence Read Archive database of the National Center of Biotechnology Information (SRP129887).Dataset 1


A major concern for transdermal drug delivery systems is the low bioavailability of targeted drugs primarily caused by the skin’s barrier function. The resistance to the carrier matrix for the diffusion and transport of drugs, however, is routinely ignored. This study reports a promising and attractive approach to reducing the resistance to drug transport in the carrier matrix, to enhance drug permeability and bioavailability via enhanced concentration-gradient of the driving force for transdermal purposes. This approach simply optimizes and reconstructs the porous channel structure of the carrier matrix, namely, poloxamer 407 (P407)-based hydrogel matrix blended with carboxymethyl cellulose sodium (CMCs). Addition of CMCs was found to distinctly improve the porous structure of the P407 matrix. The pore size approximated to normal distribution as CMCs were added and the fraction of pore number was increased by over tenfold. Transdermal studies showed that P407/CMCs saw a significant increase in drug permeability across the skin. This suggests that P407/CMC with improved porous structure exhibits a feasible and promising way for the development of transdermal therapy with high permeability and bioavailability, thereby avoiding or reducing use of any chemical enhancers. The transdermal drug delivery system has made considerable progress since the first transdermal drug patch that delivers scopolamine to treat motion sickness was approved for use in 1979 ,3. SigniThe major challenge in transdermal drug delivery consists in the skin’s outermost layer, namely, stratum corneum, which is 10–20 μm thick and has an organized structure . To enhaAdditionally, more efforts have been made to develop new transdermal techniques and construct new formulation systems, such as smart hydrogel matrix  and noveHydrogels are hydrophilic three-dimensional polymeric networks, which can absorb a large amount of water or biological fluids . The higMW = 11,500, PEO101-PPO56-PEO101) and carboxymethyl cellulose sodium (CMCs) were obtained from International Laboratory  and Wako Chemicals , respectively. Dulbecco’s phosphate-buffered saline (DPBS) was provided by Thermo Fisher Scientific . Cortex Moutan (CM) aqueous extract was a gift from the Institute of Chinese Medicine (ICM), the Chinese University of Hong Kong. Water-soluble azone (laurocapram) was purchased from Guangzhou Nanjia Chemical Technology Co. Ltd. . All other reagents were of analytical grade and used as needed.Poloxamer 407  under constant magnetic stirring. The dispersion was then refrigerated until P407 was completely dissolved. The three batches were labeled as PC160, PC200 and PC240. P407/CMCs composite hydrogel was fabricated simply by adding CMCs into P407 solution  under constant stirring with the final CMCs concentrations at 2% and 4% (w/w) labeled as PC202 and PC204, respectively. The fabrication of CM loaded composite hydrogel was similar, simply by dissolving P407 in CM solution, and the concentration of CM was 15% (w/w). The concentration of azone in the samples involved was 3% (w/w).P407/CMCs composite hydrogel was fabricated using “cold method” as described by Schmolka . BrieflyTgel).Viscosity of P407/CMCs hydrogel was analyzed using a Physica MCR301 rheometer , operated with parallel plates (diameter 25 mm). Silicon oil was used to avoid water evaporation on the outer edge of the sample. The testing temperature ranged from 5 to 40 °C with a constant heating rate of 1 °C/min at a fixed frequency of 1 Hz. The curves of complex viscosity (η*) versus temperature were plotted, the inflection points of which were indicative of the gelation transition temperature . The samples were fixed with conductive tape on a metal stub, sputter coated with gold under vacuum and observed at a 3 kV accelerating voltage at a working distance of 8.0 mm using a JEOL 6490 .The most probable pore size, total surface area, pore number fraction and pore size distribution of vacuum lyophilized P407/CMCs composite hydrogel were determined by mercury intrusion porosimetry . Around 0.2 g of each sample was placed into the sample holder cell and was penetrated by mercury at high pressure. The data were obtained using Quantachrome PoreMaster for Windows, version 8.01 .Effects of the porous structure of P407/CMCs composite hydrogel on the drug’s percutaneous behavior were comparatively analyzed using a Franz diffusion cell (receptor compartment: 6.5 mL). The porcine ear skin was selected as permeation membrane to evaluate the drug’s percutaneous behavior. Fresh skin was excised and isolated from the porcine ears  with a scalpel. The excised ear skin was then placed between the donor and the receptor compartment of the diffusion cell. The samples were positioned over the skin and covered with paraffin film. The receptor compartment of the diffusion cell was filled with DPBS (pH 7.4) with temperature at 37 ± 0.5 °C and magnetic agitation at 300 rpm. Two milliliters of the samples were withdrawn at each time interval ranging from 24 to 48 h and replenished immediately with an equal volume of fresh DPBS to maintain sink condition. One of the principal active ingredients of CM, gallic acid (GA) permeated across the skin was assayed by a high-performance liquid chromatograph  . All thePapp, cm/s) was calculated according to Equation (1) [V: the volume of the receiving chamber (6.5 cm3);A: the area of the skin surface exposed to the receiving chamber (2.8 cm2);Ci: the initial concentration of GA in the hydrogel formulation (μg/cm3);Ca/dt: the change in the concentration of GA in the receiving chamber.dThe drug’s percutaneous behavior was studied by linear regression analysis. The apparent permeability coefficient (tion (1) ,27.PappPapp value, to compare hydrogel Formulations (2) and (1), the enhancement ratio (ER) of drug permeation across the porcine skin was calculated according to Equation (2).Based on the p-value of 0.05 was considered as the required level of significance and the data were labeled with an asterisk (*) for p < 0.05, (**) p < 0.01. Each experiment was conducted thrice (n = 3).The experimental data were analyzed using Origin software , to obtain standard deviation, one-way Analysis of Variance (ANOVA) test and Bonferroni test. A w/w) and P407/CMCs composite hydrogel matrix have been discussed in our previous work [Rheological properties of P407 matrix with varying concentrations of 16%, 20% and 24%  was selected for further study. As can be seen from Based on the viscosity of P407 matrix, concentration of 20% (The pore morphology of P407 matrix with varied concentrations is shown in w/w) CMCs gave rise to the optimal pore size distribution.The surface morphology for the porous structure of P407/CMCs composite matrix a,b showsp < 0.05). By comparing the P407 matrix with different concentrations, i.e., PC160, PC200 and PC240, matrix concentration showed an appreciable influence on the percutaneous diffusivity for the model drug. A higher P407 concentration tends to result in a lower GA penetration level. This is in good agreement with the apparent permeability coefficients  shown in Papp value among the three specimens. With the presence of CMCs, both PC202 and PC204 saw a considerable increase in the transdermal diffusivity and showed higher Papp values   a. This psmallest . TherefoPapp value in p > 0.05) in the most probable pore size among the three specimens, the pore number fractions for both PC202 and PC204 were increased over tenfold compared to PC200 . A similar situation was also seen in PC204 . This suggests that the permeation behavior of model drug across the skin can be appreciably increased with the aid of azone. However, it is worth noting that, the permeability enhancement for the PC240 with azone was still far lower than that of PC204 without azone, notwithstanding that viscosity for both was almost equal. The primary cause of this phenomenon is the improved porous structure of PC204. This provides the strongest evidence for our proposal that the permeability behavior was considerably enhanced by optimizing and reconstructing the porous structure of P407 matrix through the addition of CMCs. Consequently, it can be concluded that, with the improved and optimized porous structure of P407/CMCs composite matrix, the transdermal permeability behavior can be further enhanced with the aid of penetration enhancers.In order to further investigate the percutaneous behavior of model drug from the various P407/CMCs composite matrix, azone, a commonly used chemical penetration enhancer, was also employed and formulated within the matrix . PC240 aIn summary, the presence of CMCs was found to optimize and improve the porous structure of P407 hydrogel matrix; such porous channel structure facilitates the percutaneous diffusional behavior of the model drug. Therefore, P407/CMCs composite hydrogel matrix with abundant porous structure may be an ideal drug carrier to load hydrophilic drugs for transdermal administration, avoiding or reducing the use of any chemical enhancers. Though the present study employed a small molecule model drug (GA), we believe that the porous P407/CMCs hydrogel matrix may also provide a feasible and promising way to load macromolecular drugs for transdermal application, especially with the aid of chemical penetration enhancers. Additionally, another advantage for this drug matrix is the controllable drug loading, simply by controlling the concentration of drugs when fabricating drug loaded hydrogel matrix.This study has developed a novel improved porous P407/CMCs hydrogel drug carrier for transdermal purposes, avoiding the use of chemical enhancers. It was shown that the addition of CMCs noticeably improves the porous structure of P407 matrix and the transdermal permeability behavior of loaded drug can be markedly increased. The improvement and optimization of the porous channel structure of P407/CMCs hydrogel matrix are accountable for the increased drug diffusivity within the matrix, leading to an increase in the drug concentration-gradient driving force. Accordingly, the percutaneous behavior of targeted drugs is improved. This also provides a feasible and promising strategy to load macromolecular drugs, such as vaccines and peptides, etc., for transdermal application with a high bioavailability, simply by optimizing the microstructure of the drug carrier.


Grape pomace is one of the major waste products generated by the wine-making process and it contains seeds and skins still rich in bioactive compounds. Skins can be separated from the seeds and valorised for the recovery of antioxidant extracts with different potential applications. The aim of this study was to evaluate the influence of the addition of an extract obtained from waste grape skins and encapsulated in maltodextrins on the antioxidant properties and sensory acceptability of whole wheat cocoa biscuits. Different levels of enrichment  were tested, and the obtained doughs and biscuits were analysed for total phenols content and antioxidant capacity . Extract addition increased the phenolic content (up to 134% increase) and antioxidant capacity (up to 244%) of both doughs and biscuits. Extract containing biscuits were also characterised by a different colour. However, oxidation stability, evaluated at accelerated temperature and oxygen conditions, was not improved by the extract incorporation. Cooking led to an average  16% decrease in phenolic content for both enriched and reference recipes. The enrichment level significantly influenced the sensory acceptability, with identification of two clusters of consumers, with one cluster preferring the biscuits with the highest enrichment level and one cluster preferring the biscuits with the lowest level. The results showed that whole wheat cacao biscuits represent an appropriate food matrix to develop pleasant novel products enriched in phenolic compounds from waste grape skins and likeable for regular biscuits consumers. Wine production is one of the most important agricultural activities in the world with an estimated 2017 production of 246.7 million of hectolitres , 4. In pDue to their wide consumption all over the world, cereal-based products have been largely studied for functionalization, also with ingredients from wine-making byproducts . Among cOnly a few studies exist in literature on the enrichment of biscuits with grape byproducts or related extracts –20: TablBased on these premises, the aim of this study was to evaluate the influence of the addition of an encapsulated waste grape skin extract on the functional properties and sensory acceptability of biscuits. Barbera (red grape) variety was kindly provided by a winery located in Northern Italy and treated as previously reported [Grape pomace of thereported , 23 to sreported : 4 mL/miThe obtained grape skin extract encapsulated into maltodextrins (GSM) was stored in a hermetically sealed plastic container and in desiccator until use and was analysed for dry matter content (oven drying at 105 ± 2°C for 24 h), total phenols content (TPC), and encapsulation efficiency.Looking for a healthy profile, after some preliminary trials, the following biscuit recipe was selected: 1000 g whole soft wheat flour , 300 g white sugar , 250 mL extra virgin olive oil , 240 mL mineral still water , 50 g bitter cocoa powder , and 16 g baking powder . All the ingredients were locally purchased. GSM enriched biscuits were prepared replacing part of sugar with the extract. Three levels of extract addition were tested: 1.2, 2.3, and 3.5% w/w on total ingredients, corresponding to 2.2%, 4.4%, and 6.5% on flour weight. The doughs were analysed for moisture content, TPC and AOC.Biscuits were made using a home kneading machine (Bonmann KM362CB), rolling and shaping the doughs as round biscuits  and baking in an electric static oven  11 min at 180°C.Biscuits were ground (Moulinex AD5686) and analysed for dry matter content (oven drying at 105 ± 2°C for 24 h), colour, TPC, AOC, oxidation stability, and sensory acceptability.∗ and b∗. Chroma C∗ and hue H(°) were calculated as reported in  to measure the Hunter scale parameters L, aorted in  and 2),,∗ and b∗The TPC of the GSM and the encapsulation efficiency (EE %) were evaluated, following literature . BrieflyTPC was evaluated according to the Folin assay and expressed as mg of GAE per 100 g of GSM by means of a calibration curve with a gallic acid standard  between 100 and 800 mg/L . The surterature  for phenABTS) by a calibration curve obtained with standard Trolox® ((±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid, Sigma-Aldrich) between 0.1 – 2.2 mM. TEAC is the ratio of mM Trolox® to mM phenols in the extract (as GAE) resulting in molTrolox®/molGAE [The same dough and biscuit extracts TPC were assessed for AOC according to the ABTS assay . AOC was®/molGAE .ABTS) were also calculated as μmolTrolox®_ABTS/gdm.Based on the TPC of the dough/biscuit, the specific antioxidant capacities related to dry matter of dough/biscuit   and exprOxidation stability was assessed using the Oxidation Test Reactor . Ground biscuits (30 g) were distributed homogeneously on the instrument sample holders and test conditions were set at 90°C and 6 bar oxygen pressure. The Induction Period (IP in min) was calculated by the instrument software.Sensory evaluations were conducted at the Food and Wine Sensory Laboratory of the University of Gastronomic Sciences , to assess the degree of acceptability of both the control and enriched biscuits (within one day from preparation). One hundred four consumers  were involved in the study, including students and staff of the university. Biscuits were individually presented in transparent plastic container (236 mL capacity), hermetically sealed, and identified with a three-digit code numbers. Samples were served at room temperature (25 ± 1°C) in randomized and balanced order among subjects. The consumers were verbally introduced to the computerised data collection procedure . General instructions required subjects to rinse their mouth before the beginning of the test and between samples. Participants firstly observed the biscuits and rated their liking for the appearance. Then, subjects smelled the sample and rated liking for odour. Successively, participants tasted each biscuit and judged liking for taste, flavour, and texture. Finally, the overall liking was evaluated. Liking for all parameters was rated on a 9-point hedonic scale ranging from ‘dislike extremely' (1) to ‘like extremely' (9). A timer on the computer screen required the subjects to respect a rest of 60 seconds between samples. Evaluations were conducted in individual booths under white light. At the end of the tasting session, subjects were asked for their frequency of consumption of biscuits .Results are reported as mean values of three replicates with their corresponding standard deviations. Data were subjected to one-way analysis of variance (ANOVA) using SPSS  statistical software to assess the influence of extract addition and cooking on the dough/biscuit properties. In case of significant difference (p < 0.01), Tukey's post hoc test was applied to discriminate means.Liking data were separately submitted to two-way mixed ANOVA models  by performing Fisher's Least Significance Difference . Overall liking data of all subjects were submitted to Principal Component Analysis (PCA), to obtain an Internal Preference Map (IPM) for explorative purposes. To better explore consumer's preference for biscuit prototypes, a visual oriented approach, based on the inspection of the loading plot, was used for subject clustering and Y-axis was set as limit between the two consumers' segments. Liking data of each cluster were separately submitted to two-way ANOVA models , by performing Fisher's LSD (p<0.05). Differences between the two clusters considering the declared frequency of consumption of biscuits were analysed by Pearson chi-square distribution. All sensory data analyses were conducted using the software SYSTAT version 13.1 .GAE/g and an EE% of 82.81 ± 1.15%. Extract addition did not significantly change the moisture content of the dough , suggesting the encapsulated extract enhances water release during cooking and, probably, a final harder consistency as sensorially found by Davidov-Pardo et al.  and PasqThe control dough showed a certain TPC probably due to the use of extra virgin olive oil and cocoa powder. Whole wheat flour may also have contributed . ConsideAnyway, GSM addition led to a measured TPC increase in the dough up to 134% for the 3.5% addition level . The % iCooking at 180°C led to an apparent average 16.49 ± 4.39% loss in TPC. However, the decrease was statistically significant only for the control biscuit (Sig. 0.035) and the 2.3% extract addition (Sig. 0.002).Literature is not univocal about the effects of baking on phenolics content which are reported to depend on several factors, such as type of product, type of phenolic compound, recipe, and heating conditions. For instance, an increase in free phenolic compounds due to degradation of conjugated polyphenolic compounds is reported by Abdel-Aal and Rabalski  in wholeThe addition of GSM also significantly increased the TEAC' of doughs and biscuits with different results depending on the applied assay. In fact, the % increases based on the ORAC results were comparable to those calculated for the TPC, while the ABTS assay gave higher variations .ABTS  were not significantly influenced by cooking  found by Spigno et al. [These results suggest a strong correlation between the specific antioxidant capacity and the TPC. In fact, TEAC was almost not significantly influenced neither by the GSM addition level, nor by the cooking process for both the ORAC and the ABTS assay . The fouo et al.  for a grABTS of the cookies with red grape pomace extract by Pasqualone et al. [DPPH values calculated from the data of Mildner-Szkudlarz et al. [ABTS or TEACORAC, reflecting the corresponding TEAC', but they confirmed the limited variation of this parameter with grape pomace enrichment.For comparison purpose, the TEAC for the works cited in e et al.  was lowez et al.  were hig∗) was significantly reduced by extract addition without influence of the enrichment level. Also, the value of (b∗) statistically decreased after extract addition with, in this case, a dose-dependent relation. Similarly, the hue (H(°)) and the colour saturation (C∗) significantly decreased with increasing percentage of extract addition. A high saturation value indicates more vivid and bright colours, while high values of H(°) are related to red tones. Similar results were reported by Hayta et al. [∗ following addition of red grape extracts or powder . This could be explained with the observed results of lower moisture content (except for the intermediate enrichment level) which might have modified the consistency. Despite differences found in colorimetric parameters , no signThe IPM obtained from PCA applied to the overall liking data is reported in For this cluster, even if the colour of the samples was evidently different, the visual aspect of the cookies did not affect neither the liking for appearance nor the overall liking, confirming the findings of Pasqualone et al.  on biscuχ2= 8.479; p=0.076). Cl1 comprises a higher proportion (36%) of strong biscuits consumers (declaring to consume biscuits “once a day” or “more than once a day”) compared to Cl2 (14%). Interestingly, the Cl who preferred the enriched biscuits is the most numerous one and the one composed by the highest percentage of consumers with a high frequency of biscuits consumption.A significant difference was observed between the two clusters in terms of frequency of biscuits consumption  of the final product. An apparent loss of almost 40% of the phenols added with the extract was already observed with the analysis of the dough, probably due to phenols binding with ingredients or to oxidation phenomena occurring during kneading. The phenols reduction due to baking was about 16% but not always significant. In the same way, the antioxidant activity of the product was not influenced by cooking. The results, together with comparison with literature, suggest encapsulation might have thermally protected the bioactive compounds during the process.Extract addition significantly modified the baked product colour, without being detrimental for consumers' acceptability.Results of consumer's test confirmed that the use of GSM to develop enriched biscuits is a promising issue. The significant preference observed for the cluster including the strongest biscuit consumers for the samples made with the highest GSM levels seems to indicate that whole wheat cacao biscuits represented an appropriate food matrix to develop pleasant novel products likeable for regular biscuits consumers.


Hibiscus sabdariffa), rosemary , clove (Syzygium aromaticum), and thyme (Thymus vulgaris) on some food pathogens and spoilage microorganisms. Agar well diffusion method has been used to determine the antimicrobial activities and minimum inhibitory concentrations (MIC) of different plant extracts against Gram-positive bacteria , Gram-negative bacteria , and one fungus . The extracts exhibited both antibacterial and antifungal activities against tested microorganisms. Ethanolic roselle extract showed significant antibacterial activity (P < 0.05) against all tested bacterial strains, while no inhibitory effect on Candida albicans (CA) was observed. Only the ethanolic extracts of clove and thyme showed antifungal effects against CA with inhibition zones ranging from 25.2 ± 1.4 to 15.8 ± 1.2 mm, respectively. Bacillus cereus (BC) appears to be the most sensitive strain to the aqueous extract of clove with a MIC of 0.315%. To enhance our understanding of antimicrobial activity mechanism of plant extracts, the changes in internal pH (pHint), and membrane potential were measured in Staphylococcus aureus (SA) and Escherichia coli (EC) cells after exposure to the plant extracts. The results indicated that the plant extracts significantly affected the cell membrane of Gram-positive and Gram-negative bacteria, as demonstrated by the decline in pHint as well as cell membrane hyperpolarization. In conclusion, plant extracts are of great value as natural antimicrobials and can use safely as food preservatives.This work aims to evaluate the antimicrobial potential of ethanolic and water extracts of roselle ( Globally, food spoilage caused by microorganisms still widely affects all types of food and causes food waste and loss, even in developed countries. It has been estimated that the yearly losses of global food reach up to 40% due to various factors including spoilage by microorganisms . BacteriEscherichia coli and other bacteria during storage of meat, juices, and milk , rosemary , clove (Syzygium aromaticum) as well as thyme (Thymus vulgaris) against seven common food pathogens and spoilage microorganisms, (3) to understand the mechanisms of action of tested plant extracts with respect to the potential disruption in the membrane of microorganisms and changes in cytoplasmic pH (pHint).The understanding of the mechanism of antimicrobial action of medicinal plants extracts is the first step in the optimal utilization of these extracts as natural antimicrobial agents to extend the shelf-life and maintain the food quality. With this goal, this work seeks (1) to compare between two extraction methods namely conventional extraction and ultrasound techniques (2) to examine the antimicrobial activity of ethanolic and water extracts of roselle , and two extraction methods  were used to evaluate their effect on the extraction yield.In this work, four plants were selected based on their traditional usage as folk medicine. The plants were purchased, in dried form, from the local market in Zhoushan, China. Table 1) was soaked in 180 ml of distilled water in a round bottom flask and heated for 30 min at 90°C, before the overnight incubation at 37°C, and 150 rpm in a shaking incubator  separately in beakers. Each beaker was placed in an ultrasonic bath  for 30 min at 53 kHz. Afterward, the two beakers were transferred to incubator shaker and kept for overnight at 37°C and 150 rpm. The supernatant was similarly processed as described in conventional extraction to get dried extracts of selected plants as described by . Dried e1X refers to the weight of extract after evaporation of solvent and 0X refers to the dry weight of the plant powder before extraction.Where Bacillus cereus 10451 (BC), Staphylococcus aureus 10786 (SA)], Gram-negative bacteria , as well as one pathogenic fungus [Candida albicans (F) 98001 (CA)]. SA, BC, and SE were purchased from China Center for Industrial Culture Collection , while EC was provided by Microbial Culture Collection Center of Guangdong . VP was purchased from American Type Culture Collection (ATCC), while PA and pathogenic fungus CA were obtained from National Centre for Medical culture collection (CMCC). The Gram-positive and Gram-negative bacteria were pre-cultured in Mueller Hinton broth (MHB) overnight in a rotary shaker at 37°C. Afterward, each strain was adjusted at a concentration of 108 cells/ml using 0.5 McFarland standard (595 nm) has been used to adjust the spore density of fungus at a final concentration of 106 spores/ml.The antimicrobial properties of plant extracts were tested against Gram-positive bacteria [standard . The funstandard . The speAgar well diffusion method was used to screen the antibacterial and antifungal activities of different solvent extracts as displayed by . One ml All tested extracts exhibited antimicrobial activity at a concentration of 20% (w/v). Therefore, this concentration was manipulated to determine their minimum inhibitory concentrations (MIC) using agar well diffusion method, and to evaluate their effectiveness in controlling food pathogens and spoilage microorganisms . Differeint) was investigated as an indicator of the antimicrobial mechanism of the plant extracts. In our experiment, the fluorescent probe employed was 59′69′ carboxyfluorescein diacetate succinimidyl ester (CFDA-SE). Cytoplasmic pH (pHint) was carried out as described by  buffer  and resuspended in the PP buffer. The number of cells was adjusted to 108 cells/ml by using 0.5 McFarland standard. Afterward, the bacterial cells were incubated at 37°C, 128 rpm for 30 min in the presence of 1.0 μM CFDA SE dye. The cells were centrifuged at 11200 × g, for 5 min and resuspended in PP buffer (pH 7), followed by addition of 10 mM glucose solution and incubated for an additional 30 min at 37°C. The cells were centrifuged at 11200 × g for 5 min, washed once and resuspended in PP buffer. Stained cells were then aliquoted into equal volumes for the control and treatment groups and the cell-free filtrate. One ml of plant extracts with a final concentration of 20% (w/v), were added to 1 ml of bacteria or cell-free filtrate. Fluorescence intensities were determined after 10 min by fluorescence spectrophotometer , using excitation wavelengths of 490 nm and emission wavelengths of 520 nm. While the excitation slits widths were 5 nm and the emission slits widths were 10 nm. For the control samples and bacteria free filtrate, 1 ml of PP buffer  was added to 1 ml of bacteria. Fluorescence for bacterial cells was determined by subtracting fluorescence of the respective cell-free filtrate from the treated or control groups.The two strains (SA and EC) have been chosen as an example of Gram-positive and Gram-negative bacteria to study the antibacterial mechanism of plant extracts. The changes in microbial cell cytoplasmic pH  and resuspended in the PP buffer to about 108 cells/ml using 0.5 McFarland standard. After that, 1.0 μM membrane potential-sensitive fluorescence probe DiBAC4 (3) was added and incubated for 30 min.DiBAC4(3) dye was used as described by  to deterStained cells were aliquoted into control, treatment groups and cell-free extracts. 1 ml of plant extract at a concentration of 20% was added to 1 ml of bacteria or cell-free filtrate. For control groups, 1 ml of PP buffer was added to 1 ml of bacteria or cell-free filtrate. Fluorescence intensities were determined by fluorescence spectrophotometer (HITACHI 4500), using an excitation wavelength of 492 nm and an emission wavelength of 518 nm. While the excitation slits widths were 5 nm and the emission slits widths were 10 nm, at room temperature 25°C. Background fluorescence resulting from the extracts added to the medium was determined.p-value < 0.05.The data represent mean of three replicates ± standard deviation (SD). Results were subjected to multiway analysis of variance, and the mean comparisons were performed by Tukey’s multiple range test using SPSS version 20.0 . Differences between means were considered significant at Table 1 summarized the extraction yield of each tested plant, which prepared by conventional or ultrasound method using water/ethanol. The extraction yield obtained by conventional method showed low percentage yield (Table 1) compared to those using ultrasound method. In ultrasound method, the yield of water extracts for all tested plants was much higher (p < 0.05) than ethanolic extracts (Table 1). The water extract of roselle (62.50 ± 0.51%) represented the highest yield among plant extracts followed by water extract of clove (32.16 ± 0.21%). Similarly, for ethanolic extract, the highest yield (22.22 ± 0.45%) was achieved with of roselle extract followed by clove extract (20.00 ± 0.55%), while, the lowest extract yield was obtained with thyme (15.85%) (Table 1). It has been reported previously that the water extract of different plants usually yields significantly higher amounts compared to ethanolic extracts of same plants , whereas water extract of roselle showed a maximum zone of inhibition against BC (17.0 ± 1.1 mm). The ethanolic extract of rosemary exhibited inhibitory effect against four of the pathogenic strains  while aqueous extract of rosemary was effective against three strains only . In the antifungal analysis, just ethanolic extract of clove and thyme had valuable results against CA with inhibition zone , respectively. It has been reported previously that the extracts from several plants such as oregano, cumin, cinnamon, sage, and other spices possessed significant (P < 0.05) antibacterial and antifungal activities against wide range of food spoilage bacteria (Gram-positive and Gram-negative), as well as yeast and mold  were exhibited by water extracts of clove and roselle against BC, respectively, as well as the ethanol extracts of clove (0.625%) against VP. As put forward by This study supports previous findings in the literature that the antimicrobial activities have a direct relation to increasing the extracts concentration (%) . Signifiint and membrane potential of Gram-positive (SA) and Gram-negative (EC) strains were determined. Significant decreased in cytoplasmic pHint (P ≤ 0.05) was observed after addition of plant extracts . Ethanolic extracts of clove and water extracts of thyme had the most substantial effect on both SA and EC strains; however, the water extracts of rosemary had the lowest impact compared to other extracts. The changes that occurred in the pHint indicate damage to the bacterial cell membrane  (a fluorescent dye). 4 (3), a fluorescent membrane potential stain. Our results showed a decrease in the fluorescence intensity of stained cells, which indicated displayed cell membrane hyperpolarization . Hyperpolarization has been suggested to be one of the primary indicators of membrane damage of bacteria cells (4 (3) has higher permeability and accumulation in cells with polarized membrane potential due to the presence of higher positive charges inside the cytoplasmic membrane  and cell wall disruption was observed in cells treated with plant extracts, suggesting a possible mechanism of antibacterial action. These findings indicate that the plant extracts tested in this study could be used as natural preservative agents in food to eliminate or control the growth of spoilage and pathogenic microorganisms.Based on the results, it can be concluded that using ultrasound method during extraction effectively improve the extraction yield. Overall, water and ethanolic extracts from selected plants possess antimicrobial activity as they could inhibit the growth of tested food pathogens and spoilage microorganisms. The ethanolic and aqueous extracts of roselle and clove had antimicrobial activity against all tested microorganisms except for the CA that was affected only by ethanolic extracts of clove and thyme. A decrease in cytoplasmic pH (pHFG is a student and was responsible for conducting the antimicrobial assays and contributed to the manuscript revisions mainly materials and methods. JL contributed to the development of the experimental design, data analysis, the manuscript draft, and proofreading. WM contributed to the experimental design, plants extraction, interpreted the results, as well as data analysis, and interpretation. JX and FC carried out the antimicrobial mechanism experiments, helped to write the manuscript. MC and SH were the lead investigators, designed the study, conducted the experiments, supervised the students, drafted the manuscript, and final proofreading.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.