Biomolecules are attractive building blocks with self-assembly ability, structural diversity, and excellent functionality for creating artificial materials. Heparin and protamine, a clinically relevant pair of biomolecules used in cardiac and vascular surgery, have been shown to coassemble into particulate polyelectrolyte complexes in vitro. The resulting heparin–protamine particles exhibit adhesive properties that enable advantageous interactions with proteins, cells, and various other substances and have been employed as functional materials for biomedical applications. In this review article, we summarize recent progress in research on the use of heparin–protamine particles as drug carriers, cell adhesives, and cell labels. Studies have demonstrated that heparin–protamine particles are potentially versatile in biomedical fields from drug delivery and regenerative medicine to plastic surgery. Biomolecules, such as proteins, nucleic acids, lipids and carbohydrates, form sophisticated assemblies via intermolecular interactions and constitute living organisms in nature. The self-assembly ability, structural diversity, and excellent functionality of biomolecules make them attractive as building blocks for creating artificial materials ,7,8,9,10Heparin and protamine are a clinically relevant pair of biomolecules. Heparin is a mixture of linear anionic polysaccharides with many sulfate groups and has been used clinically as an anticoagulant for more than 70 years ,21,22,23Studies have explored the in vitro coassembly of heparin and protamine and demonstrated their coassembly into nanometer- or micrometer-sized particles that are useful for biomedical applications ,31,32,33As stated above, heparin–protamine particles are useful as carriers for proteins due to their ability to adsorb proteins via electrostatic interactions and other intermolecular interactions. The particles can preserve loaded proteins from degradation by protease and from heat inactivation . MoreoveThe potential of heparin–protamine particles as drug carriers has been investigated, especially for fibroblast growth factor (FGF)-2, a protein that stimulates cell proliferation and is used clinically in wound care ,50,51. FFGF-2-containing heparin–protamine nanoparticles were used for the treatment of crush syndrome . A rat mFGF-2-containing heparin–protamine nanoparticles were shown to promote hair growth in a clinical study  56]. Tw. Tw56]. Heparin–protamine particles can carry not only FGF-2 but also other proteins. In fact, various growth factors contained in platelet-rich plasma were loaded into heparin–protamine particles . NotablyThe nonchemical modification of heparin–protamine particles has been demonstrated to be a promising strategy for creating advanced drug carriers. Nonchemical modification strategies are generally advantageous in terms of simplicity compared with the chemical modification strategies described in the next subsection. Additionally, the use of US Food and Drug Administration (FDA)-approved and clinically used drugs  may contribute to shortening the time required for safety evaluation, even for off-label use.Heparin–protamine complexes tend to have a net negative charge because anionic heparin is the major component, as stated above. The net negative charge was exploited for the nonchemical modification of particles through electrostatic interaction with a cationic peptide, GRKKRRQRRRPPQ  36]. Th. Th36]. 3) was incorporated into heparin–protamine complexes . Th. Th82]. Skin grafting is a common technique for treating burns, chronic ulcers, and skin defects after cutaneous surgical procedures ,84,85. NHeparin–protamine complexes were reported to facilitate cell labeling with iron oxide nanoparticles as described below. This cell labeling method found applications in the magnetic resonance imaging (MRI) and magnetic targeting of transplanted cells.Cell-based therapies have attracted considerable attention in regenerative medicine ,88. To uAfter pioneering work, fundamental and preclinical studies have been conducted on the clinical use of the heparin–protamine–ferumoxytol cell labeling system for MRI ,93,94,95Stem cell transplantation is a promising therapeutic strategy for acute or chronic ischemic cardiomyopathy . NeverthThis review summarizes recent research progress on heparin–protamine particles for drug carriers, cell adhesives, and cell labels . One of Despite recent progress, there is still plenty of room for further research into heparin–protamine particles. Their fundamental characteristics, including their internal structure, interaction with proteins and other substances, physicochemical and biological stability, and pharmacokinetics, have yet to be fully revealed. Furthermore, the modification of heparin–protamine particles is still in the early stages of research. We are especially interested in nonchemical modification with poly- and oligoelectrolytes, which offer a large variety of functionalities and can be readily incorporated into polyelectrolyte complexes via electrostatic adsorption. Attractive candidates include synthetic aptamers  and peptIt appears that heparin–protamine particles have been developed mostly in biomedical engineering and medicine. Nevertheless, further investigations from the perspective of molecular self-assembly may lead to innovations in assembled heparin–protamine materials. It is hoped that this review will inspire polymer and materials scientists to contribute to developing advanced biomedical materials via heparin–protamine coassembly.


Varicella zoster virus (VZV) can remain lifelong in the latent state in ganglionic neurons and adrenal glands after the initial infection. However, it can be reactivated anytime and can trigger several severe neurological manifestations such as encephalitis, meningitis, Ramsay-Hunt syndrome, cerebellitis, myelitis, and stroke. In addition, due to the diversity of clinical manifestations, clinical diagnosis of VZV can be difficult, especially in the absence of varicella. Here, we describe the case of a 52-year-old male who presented with symptoms of acute myelitis as well as polycranial neuritis, and was finally diagnosed with VZV infection through metagenomic next-generation sequencing (mNGS).A 52-year-old male came to our hospital with complaint of headache, fever, weakness of right lower limb, abdominal distension, and hearing loss. T2-weighted MRI revealed a hyperintense lesion in the spinal cord extending from T8 to T11. In addition, enhanced MRI showed small patches and strips hyperintensities in both the spinal cord and meninges. Plain abdominal radiographs and abdominal computed tomography (CT) scan displayed air-fluid levels and incomplete bowel obstruction. Moreover, electrophysiological evaluation of the peripheral neuropathy in the extremities was found to be normal. Finally, by using metagenomic next-generation sequencing (mNGS) we found that the copy number of VZV DNA in cerebrospinal fluid (CSF) was significantly increased and IgG antibody against VZV in CSF was also noted to be positive. Hence, VZV infection was identified in patient’s central neuron system. Finally, after a few days of low dose steroid treatment, the patient's symptoms were found to be significantly improved.The findings indicate that we should pay proper attention to the various symptoms caused by VZV infection due to the clinical heterogeneity, especially in the absence of cutaneous lesions. Varicella Zoster Virus (VZV) is a neurotropic DNA virus. Varicella (chickenpox) is the usual initial symptom, and then VZV can exist in a latent state in the ganglionic neurons and adrenal glands . In adulA few previous studies have been conducted on the global epidemiology of the neurological manifestations in VZV but the findings remain unclear. The various current studies are mainly regional studies or case reports. PHN is considered to be the most common complication of VZV, which can occur in ~ 15% of cases, and diagnosed in 40% of the patients over 60 years of age , 4. VZV A 52-year-old man was admitted to our hospital for headache, fever, weakness of the right lower limb, abdominal distension, and hearing loss. He was generally in good health without any medical history before. Initially, the patient experienced headache and fever, with a maximum temperature of 39 °C. Two days later, he also developed bilateral facial paralysis, right leg weakness, and bilateral hearing loss. Thereafter, two more days later, new symptoms including that of abdominal distention and urinary retention occurred. He did not experience any rash.On physical examination, he was found to be alert and oriented. The right pupil was dilated (3 mm VS. 2.5 mm) but the direct and indirect light reflection was slow, without ocular movement disorder, thereby indicating incomplete injuries of the right oculomotor nerve. Bilateral peripheral facial paralysis and presence of hearing loss suggested that his bilateral facial nerves and vestibulocochlear nerves were affected. In addition, the strength of his right lower limb appeared to be significantly weak  grade 3/5), while the left lower limb and both upper limbs were found to be normal ( (MRC) grade 5/5). It was observed that though bilateral ankle and knee jerk reflexes disappeared, whereas Babinski’s sign was positive on the right side. Furthermore, the puncture sensation below the T6 level on the left side of the body decreased markedly, while the sensation of vibration and touch were found to be normal. In addition, combined with abdominal distention and urinary retention, it was suspected that the patient suffered from a condition with spinal cord involvement.3 , of which lymphocytes accounted for 99%, and a slightly elevated protein levels to 199 mg/dl . Moreover, CSF cytology revealed primarily a lymphocyte reaction. Lyme titer, India ink capsule staining and interferon-gamma Release Assay (IGRA) were all found to be negative. Brain MRI showed mild non-specific white matter changes and cranial nerve MRI was found to be normal, with no enhancement signal in the contrast-enhanced scans   caused by VZV have also been described before , 11. MorIn our case, the patient displayed intestinal as well as urine retention, and the physical examination found crossed spinothalamic loss on one side without the posterior column loss on the opposite side, and a hyperintense T2 lesion in the spinal cord extending from T8 to T11 in enhanced spinal MRI  was initially applied in this case. VZV has been found to be a causative factor for a broad spectrum of neurological diseases , 7. HoweVZV infection may be fatal, and hence early as well as accurate diagnosis and treatment can reduce both the associated disability and mortality. We should pay proper attention to the various symptoms caused by VZV infection due to the clinical heterogeneity, especially in the absence of cutaneous lesions. Hence, comprehensive examinations should be carried out to lower misdiagnosis or missed diagnosis and provide suitable treatment to the patients.


Venous tumour thrombosis refers to the invasion of tumour into the venous system. Extramural venous invasion is routinely searched for and reported in rectal carcinoma due to its prognostic significance and influence on staging, prognosis and treatment approach. We describe a case of extramural venous invasion occurring as superior mesenteric vein tumour thrombus in the setting of a caecal carcinoma. Preliminary blood tests revealed elevated lactate at 3.8 mmol lIn the context of the non-specific abdominal tenderness and Hb drop, a contrast-enhanced CT abdomen and pelvis was performed which did not reveal any acute intra-abdominal pathology. There was also no evidence of bowel oedema or ischaemia. The patient was being managed by her general practitioner for iron-deficiency anaemia with regular iron transfusions. After overnight observation, the patient was asymptomatic and discharged home.−1. A digital rectal examination revealed melena. B12, folate studies and pelvic ultrasound were ordered and were normal. Iron studies revealed ferritin was at the lower limits of normal at 37 g l−1 (reference range 30–200), low iron of 3 mol l−1 (reference range 9.0–30.4 mol l−1), transferrin 2.7 g l−1 (reference range 2.0–3.6) and very low transferrin saturation of 4% (reference range 20–50%). A CT scan was not performed during this admission. The patient was referred to gastroenterology with a plan for outpatient colonoscopy.Around 2 months after this, the patient represented to the ED in an intoxicated state complaining of abdominal pain with Hb 80 g lInitial colonoscopy booking was not attended due to complicated psychosocial factors and over this period, there were multiple repeated emergency presentations for alcohol intoxication and symptomatic anaemia.A subsequent colonoscopy and biopsy performed 4 months after the initial ED presentation revealed a non-obstructing caecal adenocarcinoma occupying one-third of the lumen circumference measuring 5 cm in length.−1 at the time of biopsy. A staging CT and FDG-PET (fludeoxyglucose-positron emission tomography) confirmed multiple enlarged para-aortic nodes and extensive tumour invasion in the superior mesenteric vein (SMV) passing the level of the caecal tumour to the origin of the portal vein with intense metabolic activity within the primary tumour (SUV (max) 22.4) and SMV (SUV (max) 11.8) (The patient’s haemoglobin declined to 80 g lx) 11.8) . The tumx) 11.8) .The patient was planned for urgent systemic chemotherapy with an aim to downsize the tumour and potential surgical resection. In the interim, the patient developed melena and worsening abdominal pain with anaemia and was administered regular iron infusions and blood transfusions. She also developed desaturations and a CT angiography of the pulmonary arteries was performed which showed bilateral subsegmental pulmonary emboli and she was managed with therapeutic Enoxaparin.Prior to commencing chemotherapy, the patient was admitted to undertake alcohol detoxication. Unfortunately, during this period and prior to initiating chemotherapy, the patient was found deceased on her couch with post-mortem examination and toxicology finding of mixed drug toxicity as the cause of death.A post-mortem CT showed that the extent of tumour thrombus was similar to the pre-mortem staging imaging of 2 months earlier; however, the degree of calcification had increased .1,2Venous tumour thrombosis can be seen in patients with abdominal malignancies such as renal cell carcinoma, adrenal cortical carcinoma, hepatocellular carcinoma, gastric or pancreatic carcinoma. The incidence of venous tumour thrombosis in colorectal cancer, however, is rare with Sato et al in 2010 reporting venous tumour thrombosis only in 1.7% (3/176) patients with advanced colorectal cancer.Accurate diagnosis of the nature of the portal vein thrombus, related to thromboembolism or tumour thrombus, is extremely important to guide management. This can avoid unnecessary anticoagulation as would be the case to manage a benign or bland thrombus.3 This case highlights a caecal adenocarcinoma with pathologically confirmed venous tumour thrombus. Given that blood clot thrombosis and tumour thrombosis both have low-attenuation; it can be difficult to distinguish on the basis of CT alone. In general, bland thrombus is non-enhancing on imaging while tumour thrombus is enhancing and heterogeneous enhancement is generally described.The reported incidence of macroscopic tumour thrombus is very low at approximately 2.8%.As in our case, FDG-PET is useful to confirm tumour thrombus which would demonstrate intense FDG activity like the primary tumour site. However, the SUV can be unreliable in small tumours such as in the vein especially when calcification exists. Increased cross-sectional size of the vessel due to expansion is a very important factor in diagnosing tumour thrombus with confidence. Direct continuity from the primary tumour to the involved veins and shared imaging characteristics is also useful in making the correct diagnosis. Bland thrombus can enhance occasionally as it begins to recanalise, and thus as such no single feature is 100% specific and these features need to be considered overall in the specific clinical scenario. In our case, it can be presumed that the anticoagulation treated the bland component of the thrombus and the remainder was tumour thrombus, and this is consistent with the post-mortem histology finding of metastatic tumour thrombus .4 Mesenchymal, lymphoid and germ cell tumours can also calcify.4 Metastatic calcification occurs in the setting of hypercalcaemia usually due to hyperparathyroidism and often secondary to renal dysfunction.4 The mucinous subtype in colorectal cancer is most likely to calcify and also carries a worse prognosis.4 Easson et al5 also noted that some of the noncalcified colorectal metastases to the liver developed calcifications during therapy; however, they could find no association with chemotherapeutic agents or response to therapy.5Calcification within tumour thrombus is rare and not commonly reported in current literature but calcification within the tumour itself has been frequently described in abdominal malignancies like colorectal and ovarian cancers.1 suggested that patients with colorectal cancer and venous tumour thrombus are generally a cohort with more aggressive primary cancer and are at greater risk of developing liver metastasis.1 In their meta-analysis, complete tumour resection was possible in 32 (74.4%) of 43 patients with colon cancer and venous tumour thrombosis.1 In addition to being associated with a poor prognosis, venous tumour invasion also has implications for planning primary tumour surgical resection and whether it is amenable to thrombectomy and grafting.6 It also influences the adjuvant chemotherapy regime and duration which likely has to be extended in the presence of venous tumour invasion.7,8 Although in our case, the patient was planned for aggressive adjuvant chemotherapy and consideration of surgery, the complex psychosocial factors and alcohol intoxication caused delays in diagnosis and management and this contributed to an unfortunate outcome.In their review of 43 cases, Otani et alThis case highlights the importance of searching for venous invasion in colonic carcinomas as a marker of aggression and metastatic potential.Closely assessing the SMV and tributaries is important on all imaging for patients with known colorectal cancer.PET/CT is useful in differentiating blood clot venous thrombus from venous tumour thrombus.SMV invasion by tumour thrombus can significantly alter the patient’s oncological and surgical management.There is a high incidence of liver metastasis with venous tumour thrombosis which can impact on the patient’s prognosis.


Anti-programmed cell death 1 (PD-1)/programmed death-ligand 1 (PD-L1) monoclonal antibodies (mAbs) show remarkable clinical anti-tumour efficacy. However, rational combinations are needed to extend the clinical benefit to primary resistant tumours. The design of such combinations requires the identification of the kinetics of critical immune cell populations in the tumour microenvironment.In this study, we compared the kinetics of immune cells in the tumour microenvironment upon treatment with immunotherapy combinations with different anti-tumour efficacies in the non-inflamed tumour model TC-1/A9. Tumour-bearing C57BL/6J mice were treated with all possible combinations of a human papillomavirus (HPV) E7 long peptide, polyinosinic–polycytidylic acid (PIC) and anti-PD-1 mAb. Tumour growth and kinetics of the relevant immune cell populations were assessed over time. The involvement of key immune cells was confirmed by depletion with mAbs and immunophenotyping with multiparametric flow cytometry.+ T lymphocytes at day 8. Depletion of either myeloid cells or CD8+ T lymphocytes diminished the anti-tumour efficacy of the combination.The maximum anti-tumour efficacy was achieved after intratumoural administration of HPV E7 long peptide and PIC combined with the systemic administration of anti-PD-1 mAb. The intratumoural immune cell kinetics of this combination was characterised by a biphasic immune response. An initial upsurge of proinflammatory myeloid cells led to a further rise in effector CD8The anti-tumour efficacy of a successful immunotherapy combination in a non-inflamed tumour model relies on an early inflammatory process that remodels the myeloid cell compartment. This relevance relies on the ability to modulate the immune system to eliminate cancer cells.2 For this purpose, the immune baseline status of tumours, regarding certain markers such as T cell infiltration, chemokine profiles, major histocompatibility complex (MHC) class I, and programmed death-ligand 1 (PD-L1) expression, is crucial.2 Indeed, these specific characteristics have been used for tumour classification into inflamed or hot tumours, and non-inflamed or cold tumours. Inflamed tumours are characterised by an abundant infiltration of effector immune cells and are sensitive to immune checkpoint (IC) inhibitors. In contrast, non-inflamed tumours, which are more resistant to immunotherapy, present low T cell infiltration and a paucity of MHC class I and PD-L1 expression, thus favouring tumour escape.3 For these cold tumours, approaches such as vaccination, radiotherapy, chemotherapy or adoptive cellular therapy need to be combined in order to obtain a clinical response.4 Indeed, the main challenge for immunotherapy is to switch the tumour phenotype from cold to hot using appropriate treatments. In that sense, according to the cancer-immunity cycle reported by Chen and Mellman,5 several specific processes, such as activation of antigen-presenting cells (APCs), abundant tumour-infiltrating T cells and presence of interferon-α (IFN-α)/interleukin-12 (IL-12), are of particular importance to achieve effective tumour regression.9 Therapeutic cancer vaccines can promote these processes, eliciting a potent T cell-mediated immune response able to recognise and eliminate cancer cells expressing specific tumour-associated antigens.10 The success of therapeutic cancer vaccines relies on the choice of an adjuvant to ensure the proper stimulation of innate immunity. Among the different adjuvants, Toll-like receptor (TLR) agonists are often used in cancer immunotherapy.13 Thus, polyinosinic–polycytidylic acid (PIC), a TLR-3 agonist, is able to induce proinflammatory immune signalling that leads to tumour regression.15 Recently, the intratumoural (i.t.) injection of cancer vaccines has been proposed as an alternative therapeutic strategy able to induce a potent anti-tumour effect.18 However, clinical results have shown that therapeutic cancer vaccines are unable to attain significant effects on overall survival. To escape from the control of anti-tumour immune responses elicited by vaccines, tumours have developed multiple immune-resistance mechanisms such as over-expression of ICs, including the programmed cell death-1 (PD-1)/PD-L1 axis.19 Blockade of this axis using anti-PD-1/PD-L1 monoclonal antibodies (mAbs) has demonstrated unprecedented clinical benefit enhancing patient outcomes and shifting the equilibrium against tumour tolerance.21However, patient stratification and personalised treatments are required to get the most from this cancer treatment strategy.22 Thus, this study aims to compare the differences in the intratumoural kinetics of critical immune cell subsets subjected to immunotherapy combinations with a range of anti-tumour effects in a poorly immunogenic mouse tumour model.The design of clinical trials of rational combinations must be based on a profound understanding of the critical immune cell populations and the intratumoural kinetics of these cells. Unfortunately, exhaustive information about how these critical populations fluctuate over time in the tumour microenvironment is scarce. Longitudinal data collection in immunocompetent mouse models can provide an adequate characterisation of the relevant events occurring before and during immunotherapy treatment.The TLR-3 agonist PIC, IFNγ and EDTA 0.5 M (pH 8) were obtained from Thermo Fisher . The synthetic peptide DWLKYKDKLKEKLKEALFPDWLKYKDKRAHYNIVTFF that contains the human papillomavirus 16 (HPV16) E7 H2-Db-restricted epitope E7 (49–57) was synthesised by GenScript . Rat anti-mouse α-PD-1 , anti-CD8α (clone 56-6.7), anti-GR-1 (clone RB6-8C5) and polyclonal rat anti-IgG2 antibody were purchased from BioXCell . Tween-20, streptavidin−peroxidase, Percoll and bovine serum albumin were bought from Sigma-Aldrich . EDTA tubes were obtained from Stardstedt . Collagenase D and DNAase I were from Roche . ACK (ammonium-chloride-potassium) buffer was purchased from Lonza . Ninety-six V-shaped wells were bought from Greiner Bio-One , 6-well plates were from Corning  and 70 µm cell strainers were obtained from BD Falcon .TM or CytoFLEX Flow Cytometers  and data processed with FlowJoTM10.6.2 . Zombie NirTM Kit was obtained from BioLegend®  and Mouse Regulatory T Cell Staining Kit was from eBioscience . The rest of the used antibodies are listed in Table Flow cytometry analyses were performed with FACSCalibur12All experiments were carried out using the murine TC-1/A9 cell line. This cell line, derived from primary mouse lung epithelial cells, expresses the E7 protein from HPV constitutively and is characterised by low MHC class I expression.2 and 37 °C in a water-saturated atmosphere. Cells were split twice per week when a confluence of 60–70% was reached. Mycoplasma was regularly tested with a luminescence assay .Cells were maintained in RPMI-1640 with GlutaMAX supplemented with foetal bovine serum [10% (v/v)], penicillin/streptomycin [1% (v/v)], Geneticin (0.4 mg/ml) and 2-mercaptoethanol (0.05 mM), all obtained from Gibco . Growth conditions were 5% of CO5) were inoculated subcutaneously in 100 µl of phosphate-buffered saline (PBS) (Gibco) on the right flank of the animal. Seven days later, when the average tumour diameter reached 5 mm, mice were randomly divided into the corresponding groups to receive each treatment. At the end of the experiment or according to the end-point criteria established by the protocol ref. 023-17, mice were sacrificed using CO2.Five-week-old female immunocompetent C57BL/6J mice supplied by Harlan  were housed in sterile plastic cages with enrichment elements and wood chips as bedding material. A maximum of six animals were allocated to each cage and were kept under standard and sterile conditions  with sterile water and standard food ad libitum, at the animal facility of the University of Navarra. Tumour cells (1 × 10PD-L1 expression measured in TC-1/A9 culture cells and tumour-infiltrating lymphocytes (TILs) characterised ex vivo were determined to characterise the baseline immunological status of the present tumour model.5 TC-1/A9 cells per well were seeded into 6-well plates in 3 ml of medium. After 24 h, cells were collected into 96-well V-shaped cell plates and incubated 15 min at 4 °C with the rat anti-PD-L1 in PBS, followed by a stain with anti-rat-IgG-AF488 at room temperature (RT) for 15 min. In a parallel plate, PD-L1 expression was induced by 48 h exposure to IFNγ (20 ng/ml).23 These cells were washed thoroughly and stained for flow cytometry analysis.Thus, to determine PD-L1 expression, 1 × 10For TIL evaluation, tumours were collected from non-treated mice sacrificed at day 10 after TC-1/A9 cell inoculation. Tumours were kept in dry ice and immediately preserved at −80 °C embedded in OCT gel. The frozen tissue was cut into 5 µm slices and stained for CD3 and CD8. Images were scanned with the Aperio CS2 .The anti-tumour efficacy was assayed in TC-1/A9 tumour-bearing mice treated with different combinations. Female mice were randomly divided into eight groups: control; monotherapies: E7 long peptide; PIC; anti-PD-1; bitherapies: E7 long peptide/PIC; E7 long peptide/anti-PD-1; PIC/anti-PD-1; triple therapy: E7 long peptide/PIC/anti-PD-1. The E7 long peptide (100 µg/mouse) and PIC (50 µg/mouse) were administered i.t. at days 7 and 14. For i.t. administration, 100 µl were slowly injected into the tumour mass at an angle of 45°. Anti-PD-1 (200 µg/mouse) was prepared in 100 µl of PBS and injected intravenously as a bolus at days 7, 10, 14 and 17, after tumour implantation.Each group received the corresponding treatment following the experimental schedule represented in Fig. L represents the length and W the width of the tumour.where 24 were applied, taking the tumour size at day 7 after cell inoculation, as the baseline for comparisons. Complete response (CR) corresponded to a tumour size reduction of 100% compared to baseline; partial response (PR), the reduction was >50%; stable disease (SD), tumour size ranged between a reduction <50% and an increment <25% compared to baseline; finally, progressive disease was associated with an increase of the baseline >25%.Tumour growth and body weight were monitored twice weekly until the maximum tumour size was reached, and the mice were then sacrificed. In the case of tumour shrinkage, animals were monitored for 3 months to check for possible tumour relapse. Other possible signs of toxicity, such as ulceration, were carefully recorded to ensure mouse welfare. In order to explore the time evolution of the different treatments, the RECIST 1.1. criteriaFemale TC-1/A9 tumour-bearing mice were divided into eight groups, using the immunotherapy combinations described above. At different time points: 3, 8 and 13 days after treatment administration, three animals per group were sacrificed for tumour collection, as shown in Fig. g for 3 min and transferred to a V-shaped plate for the staining of CD45, CD3/TCRβ, CD8, CD11b, LY6C, PD-L1 and CD4, CD25 and Foxp3 (Table Tumour tissue was digested with 5 ml of collagenase/DNase I (1:10) for 30 min at 37 °C. The reaction was stopped by adding 50 µl of EDTA (0.5 M). Cells were forced through 70 µm cell strainers, centrifuged and incubated with 1 ml of ACK buffer for 1 min at RT. Pellets were collected by centrifugation at 600 × p3 Table . These s+ and CD8+ staining.In addition, an extra group of TC-1/A9 tumour-bearing mice injected with the triple combination was used to evaluate TILs at days 3 and 8 after treatment. Three mice at each time were sacrificed, and tumours were excised and frozen in OCT for CD3+ T cells in the anti-tumour effect of the triple therapy was investigated. Thus, at day 7 after TC-1/A9 cell inoculation, mice were divided into three groups: control and triple therapy combined with anti-GR-1 antibody or with an irrelevant antibody. The GR-1 antibody is reported to bind specifically to immature myeloid cells, leading to their depletion,25 whereas the irrelevant polyclonal rat α-IgG2 was administered as an antibody control. Anti-GR-1 and anti-IgG2 were i.p. administered at 200 µg/mouse, 24 h after treatment, at days 8 and 15. Tumour size, measured twice a week and the survival rate was used to evaluate the effect of the depletion.The role of myeloid and CD8+ T cells on the anti-tumour effect of the triple therapy, the following experiment was carried out. At day 7 after TC-1/A9 cell inoculation, mice were divided into three groups: control, triple therapy combined with anti-CD8 antibody and triple therapy combined with the irrelevant polyclonal rat anti-IgG2.To evaluate the role of CD8+ T lymphocyte depletion was confirmed in blood 24 h after anti-CD8 mAb at days 9 and 16 by flow cytometry . Groups were compared according to the type of treatment and the sampling times applying a one-way analysis of variance (ANOVA) or two-way ANOVA test, followed by multiple comparisons with the Tukey’s test. Statistical analysis of survival data was performed using RStudio (version 3.6.3) with the Survfit function from the survival package that computes the Kaplan–Meier estimator for truncated and/or censored data. The log-rank test was used to compare the different groups of treatments. Statistical significance was set to 0.05.PD-L1 is the best clinically available biomarker to predict the response to anti-PD-1/PD-L1 mAbs. Thus, we characterised the PD-L1 expression on the TC-1/A9 cell line. Flow cytometry analysis showed a low baseline expression of PD-L1 with <10% of cells being positive Fig. . HoweverNext, we assessed the TILs and PD-L1 levels and distribution in tumour sections. Immunohistochemistry analysis revealed very low TIL infiltration, mostly at the tumour margin Fig. . In lineNext, we developed different immunotherapy combinations with a wide range of response rates to set up an experimental model that allowed us to identify the immune response that characterises successful immunotherapy. The immunotherapies tested were combinations of an E7 long peptide, PIC and anti-PD-1. Figure + T lymphocytes (Fig. + myeloid cells (Fig. + T lymphocytes (Fig. + T cells at day 3 and an increase of PD-L1 expression in monocytes on day 8. However, these effects were rapidly downregulated, leading to a significant increase in Tregs on day 13. This was also the most prominent effect of PIC in monotherapy, while the anti-PD-1-alone activity was reflected in a peak expression of PD-L1 in myeloid cells on day 3.In order to identify the immune response that conveys maximum anti-tumour activity, we analysed the kinetics of different immune cells in the tumour microenvironment upon different treatments. Figure tes Fig. , Ly6C anlls Fig. , CD4+ T tes Fig.  and T-retes Fig. . The imm+ and PD-L1 in CD11b+ myeloid cells on day 3, suggesting an initial proinflammatory process. Only the combination of E7 long peptide/PIC induced an increase in CD8+ T lymphocytes and CD4+ T lymphocytes at day 8, but this effect was transient and returned to baseline levels at day 13 (Fig. + and CD4+ T lymphocytes at days 8 and 13. Despite the high increase of CD4+ T cells, Treg decreased significantly (Fig. + myeloid cells.Two-to-two combinations exerted a more profound modulation of immune cells in the tumour microenvironment. The three groups displayed a significant increase in Ly6C 13 Fig. . In conttly Fig. . The modInterestingly, those combinations achieving anti-tumour efficacy induced immune memory in those mice that completely eradicated the tumour because tumour cells were rejected after a re-challenge Fig. .+ T lymphocytes mediated by the triple therapy, we analysed the role of these cells using depleting mAbs.Based on the strong modulation of myeloid cells and CD8+ myeloid cells in the tumour microenvironment with no effect on CD8+ or CD4+ T lymphocytes (Fig. Administration of anti-GR-1 mAb 24 h after initiation of triple therapy reduced the anti-tumour efficacy Fig. . The anttes Fig. .Fig. 4Ef+ T cell-dependent anti-tumour efficacy, the antigen specificity of tumour-infiltrating CD8+ T cells was assessed by E7 tetramer staining. The number of E7-specific CD8+ T lymphocytes increased in the tumour microenvironment with those therapies that increased the total number of CD8+ T lymphocytes: E7 long peptide, E7 long peptide/PIC and triple therapy (Fig. + and CD8+ T lymphocytes 8 days after treatment (Fig. + T cells abrogated the anti-tumour effect of the combination, demonstrating the essential role these cells play in the anti-tumour response of the triple therapy (Fig. In order to evaluate the quality of CD8apy Fig. . At day apy Fig. . The maxent Fig. . Depletiapy Fig. .Fig. 5EvDue to the relevance of myeloid cells in the therapeutic efficacy of the triple therapy, we performed a detailed characterisation of myeloid-derived cells at days 1, 3 and 13 after the administration of immunotherapy combinations that exerted an anti-tumour effect: E7 long peptide/PIC, E7 long peptide/anti-PD-1 and triple therapy. Figure 27 To improve the clinical outcome of IC inhibitors, multiple combinations are being tested in clinical trials. In September 2019, 2975 combination clinical trials were active worldwide.21 As a result of this extensive clinical research, some combinations have gained approval by regulatory agencies.32 The design of these combination trials will benefit from the development of mechanistic computational models that integrate the dynamics of immune cells promoted by the treatments.35 The building of these complex models requires longitudinal data on the anti-tumour effect and effector immune cells. Thus, we designed this study to gain insight into the immune dynamics in the tumour microenvironment that characterise successful immunotherapy combinations. To achieve different anti-tumour efficacy rates, we combined the i.t. administration of the TC-1/A9-immunodominant E7 epitope in the form of a long peptide and/or the TLR-3-ligand PIC with systemic administration of the IC inhibitor anti-PD-1 mAb. Intratumoural immunotherapy can achieve a high local concentration of the immunostimulatory compounds, minimising systemic exposure and, therefore, immune-related adverse effects.36 This strategy is being pursued in clinical trials with several compounds that activate pattern recognition receptors such as TLR ligands and viruses, but intratumoural vaccination has also demonstrated clinical advantages.40 Direct inoculation of the vaccine into the tumour bed improves the activation and antigen uptake by Batf3-dependent dendritic cells, a subset of antigen-presenting cells critical for the activation of an anti-tumour immune response due to their ability to produce high levels of IL-12 in the tumour microenvironment and to cross-present tumour-associated antigens.42 In our experimental setting, the intratumoural administration of the E7 long peptide was the key immunostimulatory compound. The priming effect was triggered by the peptide alone or the peptide combined with PIC, upregulated proinflammatory markers in myeloid cells and increased the early infiltration of CD8+ T lymphocytes. In contrast, PIC or anti-PD-1 as monotherapy or the combination of both immunostimulatory compounds only modulated myeloid cells, but did not have any impact on intratumoural CD8+ T cells. Moreover, PIC markedly upregulated Treg at the latest time point. Consistent with our data, a previous study showed high levels of IL-10 production in 24 h cultures of splenocytes exposed to PIC, a phenomenon that was associated with a limited expansion and function of effector CD8+ T cells in a bacterial infection mouse model.43 The immunomodulatory effects exerted by the combination of the E7 long peptide with PIC were enhanced by the systemic administration of anti-PD-1. This triple therapy was extremely efficient at remodelling the myeloid cell compartment as early as 24 h after treatment initiation. Proinflammatory monocytes, characterised by the expression of Ly6C, were markedly increased while immunosuppressive myeloid subsets such as M2 macrophages and myeloid-derived suppressor cells decreased. This modulation of the myeloid cell compartment may be due to the release of proinflammatory cytokines in the tumour microenvironment,44 but also supports the effect of anti-PD-1 on myeloid cells as has been recently demonstrated by genetic deletion of PD-1 in myeloid cells.45 Recently, high serum levels of IL-8 released by myeloid cells in the tumour microenvironment have been shown to correlate with reduced clinical benefit to anti-PD-1/PD-L1 mAbs.47 Our data indicate that the deleterious effect of myeloid cells on IC blockade anti-tumour efficacy can be overcome by intratumoural administration of immunostimulatory agents. Modulation of myeloid cells may be superior to other strategies in clinical development aimed at depleting myeloid cells since the early modulation of myeloid cells preceded the sustained infiltration of CD8+ and CD4+ T lymphocytes, which was essential for anti-tumour efficacy. This dynamic behaviour of the critical immune subset highlights the need for preclinical-driven longitudinal sampling in biomarker studies in clinical trials. The integration of longitudinal preclinical data into mathematical computational models will help to guide new clinical trial designs.The clinical success of IC inhibitors relies on the unprecedented long-lasting responses achieved in a fraction of patients with certain tumours, such as melanoma, kidney cancer, non-small cell lung cancer and other immune-sensitive tumours.In conclusion, the analysis of the kinetics of the immune response induced by immunotherapies with different anti-tumour efficacies has revealed the critical role of the early modulation of the myeloid cell compartment to allow subsequent infiltration of effector T lymphocytes, thus highlighting the relevance of the myeloid subset as an interesting but dynamic biomarker for immunotherapies.Supplementar material


Aleurodicus rugioperculatus Martin (Hemiptera: Aleyrodidae), was documented on coconut for the first time in India. Instantaneously, RSW has garnered wide attention owing to its damage severity and rapid spread across the coconut-growing regions of the country. Hence, an attempt was made to devise a sustainable integrated pest management (IPM) module using biological control agents as a mainstay component. The present study documented the identification and characterization of a potential entomopathogenic fungal isolate for the management of RSW. An entomopathogenic fungus isolated from nymphal cadavers of RSW was identified as Simplicillium lanosoniveum based on morphological and phylogenetic analyses. A gradient of five conidial concentrations  of the S.lanosoniveum were tested against eggs, first instars, second to third instars and pupae of RSW. Results revealed that S.lanosoniveum is highly virulent to all developmental stages of RSW by causing mortality rates of 95.20%, 87.33%, 85.38% and 72.85%, in eggs, initial, middle and later instar nymphs of RSW, respectively, at the highest tested concentration (1 × 108 conidia/mL) at seven days after exposure. The LC50 and LT50 values of S.lanosoniveum were 4.72 × 104, 4.94 × 104, 5.11 × 105, 5.92 × 105 conidia/mL and 4.27, 4.86, 4.56, 5.89 days against eggs, initial, middle and later instar nymphs of RSW, respectively. Further, preliminary field trials with S.lanosoniveum strain at 1 × 108 conidia/mL exhibited a significant reduction in the egg and nymphal population by 57.8% and 56.3%, respectively. This report thus demonstrated that the newly isolated S.lanosoniveum is an effective pathogen at suppressing all the developmental stages of RSW. This is the first record of S.lanosoniveum infecting RSW, and it has a great potential to be developed as a mycoinsecticide.In 2016, infestation of an exotic polyphagous pest, the rugose spiraling whitefly (RSW),  Aleurodicus rugioperculatus Martin (Hemiptera: Aleyrodidae) is an introduced pest, whose infestation on coconut was reported for the first time from Pollachi, Tamil Nadu  and Palakkad, Kerala  state in India . RSBeauveria bassiana (Vuillemin),Akanthomyces lecanii (Zimm.) and Cordyceps fumosorosea (Wize) [Among the various biocontrol agents, entomopathogenic fungi (EPF) have been found to be successfully developed as mycopesticides in managing piercing—sucking groups of insects including whiteflies. Whiteflies are known to be infected by more than 20 species of entomopathogenic fungi, of which some of the widely studied fungi are a (Wize) ,10,11,12a (Wize) . S.lanosoniveum  on RSW. Furthermore, the efficacy of the indigenous fungal strain was investigated under laboratory and field bioassays in order to develop a biopesticide for the management of this emerging pest. Severe infestation of RSW in palm oil resulted in a reduction of 20–25% bunch yield at Andhra Pradesh . LikewisRSW adults were collected from infested coconut gardens of the research farm belonging to ICAR—Central Plantation Crops Research Institute (ICAR—CPCRI), Kasaragod, Kerala, India . RSW culture was reared on coconut seedlings raised in pots placed in the greenhouse with natural photoperiodic conditions. Different developmental stages of RSW such as eggs, first instar, second instar, third instar and fourth instar nymphs (pupae) were collected from the reared culture and were used for bioassay studies. n = 4) fully covered with fungal mycelia under natural growth conditions were collected from a coconut garden at ICAR—CPCRI, Kasaragod . Sterile needles were used to isolate mycelia or conidia from the cuticles of nymphal cadavers transferred to a Petri plate containing Potato Dextrose Agar (PDA) medium and cultured at 26 ± 1 °C and 60% RH for 7–10 days in a biochemical incubator (BOD). In order to isolate the pure culture, the hyphal tip from a small fungal colony was transferred to PDA slants and incubated at 26 ± 1 °C and 60% RH. The morpho-taxonomic characteristics of the fungus were identified based on conidia—forming mycelia and conidial structure [RSW nymphal cadavers  attached with a digital camera. The identification of the fungus was further confirmed with the help of Fungal Identification Services, Agharkar Research Institute (ARI), Pune, Maharashtra, India. viz., elongation factor-1 alpha (TEF1-α) and RNA polymerase II largest subunit 1 (RPB1) of the ribosomal RNA gene were used in addition to confirm the identification the species level. The DNA was amplified using specific primers of the TEF1-α (983F 5′ GCYCCYGGHCAYCGTGAYTTYAT 3′ 2218R 5′ ATGACACCRACRGCRACRGTYTG and RPB1 (CRPB1 5′ CCWGGYTTYATCAAGAARGT 3′ and CRPB1 A 5′ CAYCCWGGYTTYATCAAGAA 3′ RPB1Cr 5′ CCNGCDATNTCRTTRTCCATRTA 3′ [TEF1-α = MW893683 and RPB1 = MW836952) and used for multi gene phylogenetic analysis. The sequences were used as query to perform BLAST search (http://blast.ncbi.nlm.nih.gov/ (accessed on 8 April 2021), and the sequence similarity studies were performed using ClustalX (1.81). The phylogenetic tree of S.lanosoniveum was constructed following maximum likelihood (ML) method using MEGA X software [Total genomic DNA was extracted from five-day old single spore cultures of fungus grown in 100 mL Potato Dextrose broth. After vacuum filtration, the mycelia were washed with sterilized distilled water and powdered using liquid nitrogen with the help of a mortar and pestle. The DNA was extracted from powdered preparations using DNeasy Plant Mini Kit  following the standard manufacturer’s instructions. The fungal ITS region was amplified using the primers: ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) and ITS6 (5′-GGAAGTAAAAGTCGTAACAAGG-3′) . Two morsoftware .4 to 1 × 108 conidial concentration/mL were tested against different developmental stages of RSW. The fungal culture was inoculated in Potato Dextrose Broth (PDB) and incubated at 26 ± 1 °C and 60% RH on a shaker for 14 days. The mycelia mat along with conidia was thoroughly mixed in a mixer grinder, and the suspension was filtered through three layers of muslin cloth, in order to get a hyphal—free conidial suspension. Conidial suspensions were prepared in sterile distilled water containing 0.01% Tween 80 and the concentration of the suspension was adjusted to different conidial concentrations using Neubauer’s improved haemocytometer under a Nikon eclipse 80i compound microscope. Five different spore concentrations ranging from 1 × 108 spores/mL were used to determine LT50 of S. lanosoniveum against RSW developmental stages.Coconut leaf bits containing 35–40 different developmental stages of RSW were collected from the greenhouse culture. Thirty leaf bits (10 cm each) were gently dipped in freshly prepared respective conidial suspensions for 20 s and placed separately on a Petri plate (14 × 2.5 cm) over sterilized wet tissue paper. Sterile distilled water containing 0.01% Tween 80 alone was used as the control. The experiment had five replications for each concentration, and the whole bioassay was repeated twice. Prior to conidial spray, the number of various developmental stages of RSW on each coconut leaf bit was counted to maintain a uniform population across the treatments. The mortality data were recorded by counting dead cadavers and developmental stages with fungal infection at 24 h intervals up to seven days, and the data was subjected to Abbott’s formula [S.lanosoniveum carried out during November 2019 and January 2020, respectively, in the RSW infested coconut gardens of ICAR—CPCRI, Kasaragod, Kerala . The conidial suspension of the fungal culture (1 × 108 conidia/mL) was prepared in sterile aqueous solution containing 0.01% Tween 80. The spore suspension of 500 mL was sprayed thoroughly on the dwarf cultivars of each coconut tree , n = 20) which were naturally infested with different developmental stages of RSW through handheld sprayer. Prior to fungal spray, the numbers of developmental stages of RSW per leaflet  were counted under a Nikon Stereozoom microscope (SMZ 800 N). Twenty dwarf palms sprayed with sterile aqueous solution containing 0.01% Tween 80 were maintained as control. Mortality data ware recorded from the treated and control plants after 14 days of spray. The experiment was replicated thrice and field bioassays were conducted twice at different time intervals. Developmental stages with S. lanosoniveum fungal infection recorded using a Nikon Stereozoom microscope (SMZ 800 N) and the percent mortality was calculated. Two field trials using A. rugioperculatus were corrected using Abbott’s formula [p < 0.05. The dose and time dependent mortality studies to kill 50% of the population (LC50 and LT50) were calculated by probit analysis [t-test with a significance of difference at p < 0.01 [Conidial concentration, time and their interactive effects on mortality per cent data were analyzed using PROC GLM . Mortality data of different developmental stages of analysis  using R analysis ,23. Fielp < 0.01 . A. rugioperculatus was identified as Simplicilliumlanosoniveum . The fungal colonies were characterized on PDA medium as white, velvety with radial cracks and primrose yellow on the reverse side . A phylogenetic tree of Simplicillium at species level was generated using maximum-likelihood (ML) analysis based on a combined data set of ITS, TEF1-α and RPB1. Multigene phylogenetic analysis showed that Simplicillium isolate  of the present study have a close phylogenetic affinity to Simplicillium lanosoniveum with strong bootstrap support , middle (second—third) and later (fourth) instar nymphs of RSW with corrected mortality rates of 95.20%, 87.33%, 85.38% and 72.85%, respectively following the seventh day after treatment (DAT) at 1 × 108 conidial concentrations/mL  while low conidial concentration (1 × 104 conidia/mL) exhibited mortality of 41.25% at 7 DAT  . Likewise, first instars experienced highest mortality (87.33%) at conidial concentrations of 108 conidia/mL  whereas lowest per cent mortality (39.64%) was observed at a dose of 104 conidia/mL on 7 DAT . Similarly, on seven DAT, the highest mortality of 85.38% was observed in second to third instar nymphs treated with conidial concentrations of 108 conidia/mL and the lowest concentration of 104 conidia/mL recorded lowest mortality of 35.38%   . Expecteon 7 DAT . As obse 0.0001) . 8 conidia/mL, whereas the lowest mortality of 24.76% was observed at the dose of 104 conidia/mL on 7 DAT . The mean mortality in pupae ranged from 1.25% to 4.33% on 4 DAT when treated with conidial concentration of 104 conidia/mL  and 108 conidia/mL , respectively . Overall, the mortality rate at various developmental stages of RSW has significantly increased with an increase in the concentration of fungal conidia and the duration of the time following the exposure to the fungal treatments. In general, significant differences in the entomopathogenic pathogenicity of S. lanosoniveum against RSW were observed between conidial concentration , time interval  and stages of host insect . Significant interactions were observed between treatment*stage of host insect , treatment*time , time*stage of host insect  and treatment*stage of host insect*time .In case of pupae, highest mortality of 72.85% was documented with the dose of 10ectively . A simil50 and LT50 values of S. lanosoniveum against different life stages of A. rugioperculatus were documented based on the dose—and time mortality response studies (50) values of S. lanosoniveum against eggs, first instars, second to third instarsand pupae of RSW were 4.72 × 104, 4.94 × 104, 5.11 × 105, 5.92 × 105 conidia/mL respectively. The LT50 values of S. lanosoniveum at the highest concentration (1 × 108 spores/mL) on the corresponding developmental stages were 4.27, 4.86, 4.56, 5.89 days, respectively  and nymphs  of A. rugioperculatus were reduced by 57.8% and 56.3%, respectively at 14 DAT. Thus, the native fungi isolated and reported herein showed positive virulence by infecting RSW stages under open field conditions (Fourteen days after the spray of nditions .Encarsia guadeloupae Viggiani (Hymenoptera: Aphelinidae) [Akanthomyces species are widely utilized as commercial biological pesticides against whiteflies, thrips and aphids [Akanthomyces were later placed in the genera Simplicillium and Lecanicillium based on systematic studies [Simplicillium occurs in a broad range of ecological niches [Simplicillium sp. against insects [S. lanosoniveum isolated from RSW in the invaded region. Bioassay studies have revealed the potency of the isolate S. lanosoniveum, as it was found to be virulent against all the developmental stages of RSW. The ecological and economical value of Simplicillium sp. for its biocontrol and bioactive features were highlighted by many researchers [S.lanosoniveum is a known phytopathogen, causing brown spots and lesions on flowers [Trichomonas vaginalis agent [Simplicillium such as S. chinense acts as a biological control agent against plant parasitic nematodes [S. lamellicola is known to suppress plant bacterial diseases and grey mould of tomato and ginseng [S. lanosoniveum [S. lamellicola and S. obclavatum [S. lanosoniveum was demonstrated against silkworms, and it was evident that virulence of the isolate was strong as that of B. bassiana [S. longisoniveum, exhibited >70–80% mortality rates against RSW nymphs and pupae, while >90% mortality recorded for egg stage at 1 × 107 and 1 × 108 conidia/mL under laboratory bioassays on seven DAT. It was noticed that early instars are highly susceptible to this fungal strain than the late instars. Present study results are corroborating the earlier report of Nagasi et al. [B. bassiana was found most virulent against the first instar of silver leaf whitefly, Bemisia argentifolii (Bellows & Perring). Entomopathogenic fungi used as an effective biological control against in other whitefly species like, B. argentifolii and Trialeurodes vaporariorum (Westwood) [B. bassiana caused 52–98% mortality in Bemisia tabaci (Gennadius) at concentrations of 1–4 × 106 conidia/mL. The present study observed increased mortality of different developmental stages of RSW with an increase in the conidial concentration and time of exposure. Likewise, Boopathi et al. [I. fumosorosea was highly pathogenic to A. dispersus on cassava and exhibited differences in efficacy between 3 and 15 days after treatment. In the present study, the LC50 values and LT50 values of S. lanosoniveum against eggs, first instar, second to third instar, and pupae of RSW were 4.72 × 104, 4.94 × 104, 5.11 × 105, 5.92 × 105 conidia/mL and 4.27, 4.86, 4.56, 5.89 days, respectively. Similarly to present results, the LC50 values of I. fumosorosea for eggs, first—second, third and fourth instar nymphs of RSW were estimated to be 2.6 ×104, 2.3 × 104, 3.5 × 105 and 9.5 × 105 spores /mL respectively and the LT50 values were4.19, 3.44, 5.62 and 5.69, respectively for corresponding life stages for the concentration of 1 × 108 conidia/mL tested [50 and LT50 values of S. lanosoniveum exhibited significant biocontrol potential in the management of RSW under laboratory conditions. The results are comparable to the effects produced by the widely studied entomopathogenic fungi like I. fumosorosea [S. longisoniveum at 1 × 108 conidia/mL tested under field conditions caused 57.8% and 56.3% mortality in the egg and nymphal population of RSW on 14 DAT, which was less than that of laboratory efficacy. The reason for this reduction in efficacy in the field condition might be due to the involvement of abiotic factors, as field conditions are heterogenetic in nature compared to the homogenic environment of the laboratory. Earlier reports also suggested that environmental factors not only affect survival and virulence of entomopathogenic fungi, but also influence their host–pathogen interaction [S. lanosoniveum on RSW. This study identified a highly pathogenic fungal isolate, S. lanosoniveum, based on its morphology, molecular and phylogeny and demonstrated its pathogenicity against RSW under in vitro and field conditions. Hence, this study forms a foundation for the large scale development of a biocontrol agent to suppress the spread of RSW. However, further studies on its compatibility with natural enemies for field applications are required. Efforts are also in progress to develop formulations with high virulent stability to sustain its field efficacy.The first incidence of the RSW was identified in India during the year 2016 following which it has spread widely and caused serious damages in larger areas of crop cultivation, particularly in coconut ,8. The alinidae) ,8. The clinidae) ,4,5,6. Hlinidae)  owing tolinidae) ,27. Amond aphids . Fungicol niches ,30,31,32 insects ,38,39,40earchers ,42,43. S flowers  or as a  flowers ,46. This flowers  and as ais agent . Other sematodes . S. lame ginseng . Bioactisoniveum ,51,52,53clavatum . The enti et al. , where, estwood) ,56,57. Eestwood)  reportedi et al.  also obsL tested . Therefoosorosea . Howevereraction . Temperaeraction ,64. In aeraction . Howevereraction . Therefo


The success of nanotechnology in the field of physical, chemical and medical sciences has started revolutionizing the drug delivery science and theranostics (therapy and diagnostics) ,2. The sIn this case of the active substance, the drug-delivery vehicle, as a critical quality attribute in the drug delivery science, needs special attention for the formulation development, which can be successfully achieved via nanotechnology. Drugs incorporated in nanocarriers, either physically entrapped or chemically tethered, have the potential to target the physiological zone of the disorder sparing normal cells from collateral consequences. Targeting several molecular mechanisms, for either treatment or prevention of difficult-to-treat diseases, for the design of various nanotechnology-based drug delivery systems is one of the prime focuses of the formulation scientist at the present juncture. Much attention has been devoted to developing new drug-delivery systems with many advantages compared with the conventional forms of dosage, such as, among others, enhanced bioavailability, greater efficiency, lower toxicity, controlled release ,10,11,12Materials designed for biomedical applications should be characterized by a high sensitivity and specificity, a lack of functional interference with the sample, photochemical stability, non-toxicity, long time of storage and, as far as possible, detection of a substance in the presence of others. Moreover, nanoscale materials have been exploited as active components in a wide range of technological applications in the biomedical field ,23,24,25It is well-known that nanoscale fluorescent materials have attracted much interest due to the increasing demand for efficient photosensitive materials not only for sophisticated optoelectronic and photonic devices but also for a broad range of biomedical applications ,32,33,3410(XO4)6Y2, where M represents divalent cations , XO4 = PO43−, VO43−, etc. and Y represents anions: F−, OH−, Cl−, Br−, etc. The hexagonal structure in apatites belongs to 3/mP6 space group and allows the cations to localize in the 4(f) and 6(h) positions [10(PO4)6(Y)2, are biocompatible and are natural building blocks for bones and teeth [Apatites are inorganic compounds with a general formula Mositions  and is aositions , allows nd teeth . This fend teeth . Howevernd teeth  which isnd teeth . 10(PO4)6(OH)2 for bio-applications [Several strategies have been developed in the synthesis of nanoparticles so far, involving such techniques as microemulsion, precipitation, thermal decomposition, chemical vapor deposition and others. However, the best results and control over particle size, crystallinity and purity can be ensured using microwave technology . For insications ,52. Anotications ) which cications . For exaications . To dateications . One of ications . 2+ in biological cells are widely studied with fluorescent probes. The probes have a high selectivity for free calcium and exhibit marked changes in their photophysical properties upon binding. In particular, changes in fluorescence intensity (intensity probes) or spectral shift (ratio probes) upon binding to Ca2+ are monitored. The main drawback of intensity probes is that the intensity of fluorescence is affected by both the probe concentration and the free Ca2+ concentration. Consequently, a quantitative determination of Ca2+ distributions requires the probes to be distributed homogeneously in the sample. Conventional quantitative determinations of Ca2+ concentration with ratio probes overcomes the dependence on local probe concentration by exploiting ratiometric procedures using excitation or detection at two wavelengths [Furthermore, calcium is the fifth most abundant element by mass in the human body 1.4–1.66%) where it is a common cellular ionic messenger with many functions and serves also as a structural element in bones (hydroxyapatites—99%) . Calciumelengths ,60. The .4–1.66% elengths ,62,63,64elengths . FluoresNanocrystalline probes doped with lanthanide ions based on apatites meet these requirements . Their n