Gli2 Facilitates Tumor Immune Evasion and Immunotherapeutic Resistance by Coordinating Wnt Ligand and Prostaglandin Signaling.

Therapeutic resistance to immune checkpoint blockade has been commonly linked to the process of mesenchymal transformation (MT) and remains a prevalent obstacle across many cancer types. An improved mechanistic understanding for MT-mediated immune evasion promises to lead to more effective combination therapeutic regimens. Herein, we identify the Hedgehog transcription factor, Gli2, as a key node of tumor-mediated immune evasion and immunotherapy resistance during MT. Mechanistic studies reveal that Gli2 generates an immunotolerant tumor microenvironment through the upregulation of Wnt ligand production and increased prostaglandin synthesis. This pathway drives the recruitment, viability, and function of granulocytic myeloid-derived suppressor cells (PMN-MDSCs) while also impairing type I conventional dendritic cell, CD8 + T cell, and NK cell functionality. Pharmacologic EP2/EP4 prostaglandin receptor inhibition and Wnt ligand inhibition each reverses a subset of these effects, while preventing primary and adaptive resistance to anti-PD-1 immunotherapy, respectively. A transcriptional Gli2 signature correlates with resistance to anti-PD-1 immunotherapy in stage IV melanoma patients, providing a translational roadmap to direct combination immunotherapeutics in the clinic. SIGNIFICANCE: Gli2-induced EMT promotes immune evasion and immunotherapeutic resistance via coordinated prostaglandin and Wnt signaling.

A SREBF2-dependent gene program drives an immunotolerant dendritic cell population during cancer progression.

Dendritic cells (cDCs) are essential mediators of anti-tumor immunity. Cancers have developed mechanisms to render DCs dysfunctional within the tumor microenvironment. Utilizing CD63 as a unique surface marker, we demonstrate that mature regulatory DCs (mregDCs) suppress DC antigen cross-presentation while driving T H 2 and regulatory T cell differentiation within tumor-draining lymph node tissues. Transcriptional and metabolic studies show that mregDC functionality is dependent upon the mevalonate biosynthetic pathway and the master transcription factor, SREBP2. Melanoma-derived lactate activates DC SREBP2 in the tumor microenvironment (TME) and drives mregDC development from conventional DCs. DC-specific genetic silencing and pharmacologic inhibition of SREBP2 promotes anti-tumor CD8 + T cell activation and suppresses melanoma progression. CD63 + mregDCs reside within the sentinel lymph nodes of melanoma patients. Collectively, this work describes a tumor-driven SREBP2-dependent program that promotes CD63 + mregDC development and function while serving as a promising therapeutic target for overcoming immune tolerance in the TME. One Sentence Summary: The metabolic transcription factor, SREBF2, regulates the development and tolerogenic function of the mregDC population within the tumor microenvironment.

Tumor-intrinsic NLRP3-HSP70-TLR4 axis drives premetastatic niche development and hyperprogression during anti-PD-1 immunotherapy.

The tumor-intrinsic NOD-, LRR- and pyrin domain-containing protein-3 (NLRP3) inflammasome-heat shock protein 70 (HSP70) signaling axis is triggered by CD8+ T cell cytotoxicity and contributes to the development of adaptive resistance to anti-programmed cell death protein 1 (PD-1) immunotherapy by recruiting granulocytic polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) into the tumor microenvironment. Here, we demonstrate that the tumor NLRP3-HSP70 axis also drives the accumulation of PMN-MDSCs into distant lung tissues in a manner that depends on lung epithelial cell Toll-like receptor 4 (TLR4) signaling, establishing a premetastatic niche that supports disease hyperprogression in response to anti-PD-1 immunotherapy. Lung epithelial HSP70-TLR4 signaling induces the downstream Wnt5a-dependent release of granulocyte colony-stimulating factor (G-CSF) and C-X-C motif chemokine ligand 5 (CXCL5), thus promoting myeloid granulopoiesis and recruitment of PMN-MDSCs into pulmonary tissues. Treatment with anti-PD-1 immunotherapy enhanced the activation of this pathway through immunologic pressure and drove disease progression in the setting of Nlrp3 amplification. Genetic and pharmacologic inhibition of NLRP3 and HSP70 blocked PMN-MDSC accumulation in the lung in response to anti-PD-1 therapy and suppressed metastatic progression in preclinical models of melanoma and breast cancer. Elevated baseline concentrations of plasma HSP70 and evidence of NLRP3 signaling activity in tumor tissue specimens correlated with the development of disease hyperprogression and inferior survival in patients with stage IV melanoma undergoing anti-PD-1 immunotherapy. Together, this work describes a pathogenic mechanism underlying the phenomenon of disease hyperprogression in melanoma and offers candidate targets and markers capable of improving the management of patients with melanoma.

Drug Sensitivity Testing for Cancer Therapy, Key Areas.

AIMS: Cancer is a high-mortality disease (9.6 million deaths in 2018 worldwide). Given various anticancer drugs, drug selection plays a key role in patient survival in clinical trials. METHODS: Drug Sensitivity Testing (DST), one of the leading drug selective systems, was widely practiced for therapeutic promotion in the clinic. Notably, DSTs assist in drug selection that benefits drug responses against cancer from 20-22% to 30-35% over the past two decades. The relationship between drug resistance in vitro and drug treatment benefits was associated with different tumor origins and subtypes. Medical theory and underlying DST mechanisms remain poorly understood until now. The study of the clinical scenario, sustainability and financial support for mechanism and technical promotions is indispensable. RESULTS: Despite the great technical advance, therapeutic prediction and drug selection by DST needs to be miniature, versatility and cost-effective in the clinic. Multi-parameters and automation of DST should be a future trend. Advanced biomedical knowledge and clinical approaches to translating oncologic profiles into drug selection were the main focuses of DST developments. With a great technical stride, the clinical architecture of the DST platform was entering higher levels (drug response testing at any stage of cancer patients and miniaturization of tumor samples). DISCUSSION: The cancer biology and pharmacology for drug selection mutually benefit the clinic. New proposals to reveal more therapeutic information and drug response prediction at genetic, molecular and omics levels should be estimated overall. CONCLUSION: By upholding this goal of non-invasive, versatility and automation, DST could save the life of several thousand annually worldwide. In this article, new insights into DST novelty and development are highlighted.

Anti-inflammatory Activity of PLA2 Inhibitory Saccharumoside-B.

BACKGROUND: Saccharumoside-B and its analogs were found to have anticancer potential in vitro. The present study reports acute toxicity, molecular docking, ADMET profile analysis, and in vitro and in vivo anti-inflammatory activity of saccharumoside-B for the first time. METHODS: The in vitro enzyme inhibitory activity of saccharumoside-B on PLA2, COX-1, COX-2, and 5-LOX enzymes was evaluated by the cell-free method, and its effect on TNF-α, IL1ß, and IL- 6 secretion levels in LPS stimulated THP-1 human monocytes was determined by ELISA-based methods. The anti-inflammatory activity was evaluated in vivo by carrageenan-induced rat paw edema model. To test its binding affinity at the active site pockets of PLA2 enzymes and assess drug-like properties, docking experiments and ADMET studies were performed. RESULTS: Saccharumoside-B showed selective inhibition of the sPLA2 enzyme (IC50 = 7.53 ± 0.232 µM), and thioetheramide-PC was used as a positive control. It showed significant inhibition (P ≤ 0.05) of TNF-α, IL-1ß, and IL-6 cytokines compared to the positive control dexamethasone. Saccharumoside-B showed a dose-dependent inhibition of carrageenan-induced rat paw edema, with a maximum inhibition (76.09 ± 0.75) observed at 3 hours after the phlogistic agent injection. Saccharumoside-B potentially binds to the active site pocket of sPLA2 crystal protein (binding energy -7.6 Kcal/Mol). It complies with Lipinski's Rule of Five, showing a promising safety profile. The bioactivity scores suggested it to be a better enzyme inhibitor. CONCLUSION: Saccharumoside-B showed significant PLA2 inhibition. It can become a potential lead molecule in synthesizing a new class of selective PLA2 inhibitors with a high safety profile in the future.

Targeting Triglyceride Metabolism for Colorectal Cancer Prevention and Therapy.

BACKGROUND: Triglycerides (TG) are one of the major constituents of body fat and energy reservoir, which consist of an ester derived from glycerol and three free fatty acids. TG lipase, monoacylglycerol lipase, fatty acid synthase, and HMG-CoA reductase are some of the key enzymes related to TG metabolism, and their roles in colorectal cancer (CRC) initiation and progression are under investigation. METHODS: The literature search was performed based on various published papers, mostly on triglyceride metabolism relevant to CRC in PubMed, Google Scholar and other search engines. The gene expression profiling of some of the TG metabolic pathway mediators was performed by transcriptomic and/or proteomic data from The Cancer Genome Atlas (TCGA) database using R program and cBioportal software. RESULTS AND DISCUSSION: Accumulating pieces of evidence suggest that TG profiling may be used as a biomarker for the diagnosis and/or prognosis of CRC. Dysregulation of TG metabolism is associated with the initiation and progression of CRC. Most of the TG anabolic pathway mediators are overexpressed and/or overactivated during CRC tumorigenesis, while most TG catabolic pathway mediators are downregulated and/or inactivated based on literature search and correlated with TCGA data. Metabolic enzymes of TG and FAs metabolic pathways are involved in CRC tumor growth survival and metastasis. CONCLUSION: Overall studies from the previous literature and our TCGA data analysis demonstrated that the area of research on TG-associated lipid metabolic pathways holds great promise and warranted detailed investigations in this area for the implementation of novel preventive and therapeutic strategies against CRC.

Features, Pharmacological Chemistry, Molecular Mechanism and Health Benefits of Lemon.

BACKGROUND: Citrus limon, a Mediterranean-grown citrus species of plants belonging to the Rutaceae family, occupies a place of an impressive range of food and medicinal uses with considerable value in the economy of the fruit of the country. Citrus fruits are economically important with large-scale production of both the fresh fruits and industrially processed products. The extracts and phytochemicals obtained from all parts of C. limon have shown immense therapeutic potential because of their anticancer, anti-tumor and anti-inflammatory nature, and also serve as an important ingredient in the formulation of several ethnic herbal medicines. These properties are mediated by the presence of different phytochemicals, vitamins and nutrients in the citrus fruits. MATERIAL AND METHODS: The methods involved in the preparation of the present article included the collection of information from various scientific databases, indexed periodicals, and search engines such as Medline Scopus google scholar PubMed, PubMed central web of science, and science direct. RESULTS: This communication presents an updated account of different pharmacological aspects of C. limon associated with its anti-oxidative, antiulcer, antihelmintic, insecticidal, anticancer, cytotoxic, and estrogenic activities. In addition, C. limon extracts possess hepatoprotective, anti-hyperglycemic, and antimicrobial properties. The present article includes the structure and function of different key chemical constituents from different parts of C. limon. Also, the possible molecular mechanisms of actions of bioactive compounds from C. limon are displayed. CONCLUSION: The traditional and ethno-medicinal literature revealed that C. limon is very effective in different pathologies. Most of these compounds possessing antioxidant properties would be implicated in offering health benefits by acting as potential nutraceuticals to humans with special reference to disease management of health and disease.

Molecular Targets in Precision Chemoprevention of Colorectal Cancer: An Update from Pre-Clinical to Clinical Trials.

Colorectal cancer (CRC) is one of the leading causes of cancer deaths worldwide. The initiation and progression of CRC is a multi-step process that proceeds via precursor lesions to carcinoma, with each stage characterized by its distinct molecular and tissue microenvironment changes. Precursor lesions of CRC, aberrant crypt foci, and adenoma exhibit drastic changes in genetic, transcriptomic, and proteomic profiles compared to normal tissue. The identification of these changes is essential and provides further validation as an initiator or promoter of CRC and, more so, as lesion-specific druggable molecular targets for the precision chemoprevention of CRC. Mutated/dysregulated signaling (adenomatous polyposis coli, ß-catenin, epidermal growth factor receptor, V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), tumor protein53, Akt, etc.), inflammatory (cyclooxygenase-2, microsomal prostaglandin E synthase-1, inducible nitric oxide synthase, and other pro-inflammatory mediators), and metabolic/growth factor (fatty acid synthase, ß-Hydroxy ß-methylglutaryl-CoA reductase, and ornithine decarboxylase) related targets are some of the well-characterized molecular targets in the precision chemoprevention of CRC. In this review, we discuss precursor-lesion specific targets of CRC and the current status of pre-clinical studies regarding clinical interventions and combinations for better efficacy and safety toward future precision clinical chemoprevention. In addition, we provide a brief discussion on the usefulness of secondary precision chemopreventive targets for tertiary precision chemoprevention to improve the disease-free and overall survival of advanced stage CRC patients.

Salidroside - Can it be a Multifunctional Drug?

BACKGROUND: Salidroside is a glucoside of tyrosol found mostly in the roots of Rhodiola spp. It exhibits diverse biological and pharmacological properties. In the last decade, enormous research is conducted to explore the medicinal properties of salidroside; this research reported many activities like anti-cancer, anti-oxidant, anti-aging, anti-diabetic, anti-depressant, anti-hyperlipidemic, anti-inflammatory, immunomodulatory, etc. Objective: Despite its multiple pharmacological effects, a comprehensive review detailing its metabolism and therapeutic activities is still missing. This review aims to provide an overview of the metabolism of salidroside, its role in alleviating different metabolic disorders, diseases and its molecular interaction with the target molecules in different conditions. This review mostly concentrates on the metabolism, biological activities and molecular pathways related to various pharmacological activities of salidroside. CONCLUSION: Salidroside is produced by a three-step pathway in the plants with tyrosol as an intermediate molecule. The molecule is biotransformed into many metabolites through phase I and II pathways. These metabolites, together with a certain amount of salidroside may be responsible for various pharmacological functions. The salidroside based inhibition of PI3k/AKT, JAK/ STAT, and MEK/ERK pathways and activation of apoptosis and autophagy are the major reasons for its anti-cancer activity. AMPK pathway modulation plays a significant role in its anti-diabetic activity. The neuroprotective activity was linked with decreased oxidative stress and increased antioxidant enzymes, Nrf2/HO-1 pathways, decreased inflammation through suppression of NF-κB pathway and PI3K/AKT pathways. These scientific findings will pave the way to clinically translate the use of salidroside as a multi-functional drug for various diseases and disorders in the near future.

Publisher Correction: Synthesis of Saccharumoside-B analogue with potential of antiproliferative and pro-apoptotic activities.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Discovery and Development of a Novel mPGES-1/5-LOX Dual Inhibitor LFA-9 for Prevention and Treatment of Chronic Inflammatory Diseases.

BACKGROUND: Non-steroidal anti-inflammatory drugs, cyclooxygenase (COX)-2 selective inhibitors, have been explored for prevention and treatment of several inflammatory chronic conditions including arthritis, and cancer. However, the long-term use of these drugs is associated with gastrointestinal, renal, and cardiovascular side effects. Later, COX/5-lipoxygenase (5-LOX) dual inhibitors (eg, licofelone) have been developed but did not enter into the market from the clinical trails due to COX-1/2 inhibition-associated side effects. Hence, targeting microsomal prostaglandin E synthase-1 (mPGES-1) and 5-LOX can be an ideal approach while sparing COX-1/2 activities for development of the next generation of anti-inflammatory drugs with better efficacy and safety. MATERIALS AND METHODS: In silico (molecular modelling) studies were used to design a mPGES-1/5-LOX dual inhibitory and COX-1/2 sparing lead molecule licofelone analogue-9 (LFA-9) by modifying the pharmacophore of licofelone. In vitro cell-free enzymatic (mPGES-1, 5-LOX, COX-1/2) assays using fluorometric/colorimetric methods and cell-based assays (LPS-induced PGE2, LTB4, and PGI2 productions from macrophages) using ELISA technique, isothermal calorimetry, and circular dichroism techniques were performed to determine the mPGES-1/5-LOX inhibitory efficacy and selectivity. Anti-inflammatory efficacy of LFA-9 was evaluated using a carrageenan (inflammogen)-induced rat paw edema model. Infiltration/expression of CD68 immune cells and TNF-α in paw tissues were evaluated using confocal microscope and immunoblot analysis. Anti-cancer effect of LFA-9 was evaluated using colon spheroids in vitro. RESULTS: LFA-9 inhibited mPGES-1/5-LOX and their products PGE2 and LTB4, spared COX-1/2 and its product PGI2. LFA-9 bound strongly with human mPGES-1/5-LOX enzymes and induced changes in their secondary structure, thereby inhibited their enzymatic activities. LFA-9 inhibited carrageenan-induced inflammation (70.4%) in rats and suppressed CD68 immune cell infiltration (P ≤ 0.0001) and TNF-α expression. LFA-9 suppressed colon tumor stemness (60.2%) in vitro through inhibition of PGE2 (82%) levels. CONCLUSION: Overall study results suggest that LFA-9 is a mPGES-1/5-LOX dual inhibitor and showed anti-inflammatory and colorectal cancer preventive activities, and warranted detailed studies.

MAPK signalling pathway in cancers: Olive products as cancer preventive and therapeutic agents.

The mitogen-activated protein kinases (MAPKs) are fundamental in inflammation and cancer control, through the crosstalk between the redox regulated nuclear factor E2-related factor 2 (Nrf2) and nuclear factor-kB (NFκB) gene expression. MAPKs regulate various cellular activities involved in cancer progression, including proliferation, apoptosis and immune escape and blockade of upstream kinases is a current therapeutic strategy. However, these therapies are associated with some adverse effects and with the paradoxical activation of the MAPKs pathway. In the context of cancer prevention and treatment, it has been suggested that dietary factors are able to modulate cancer initiation and progression by interacting with the MAPKs. Within these dietary factors, virgin olive oil (VOO) is of particular interest due to its content in squalene, already used as drug delivery system in cancer therapy. The aim of this review is to discuss the studies pointing to the effects of olive-derived foodstuff and nutraceuticals on MAPKs signalling cascades. The reviewed experimental studies suggest that the stress-activated JNK and p38 MAPKs could be targets of olive-derived nutraceuticals. The latter, including phytochemicals from olive cultivation and processing wastes, could be adjuvants in chemotherapies, whereas VOO could be considered a "natural delivery system" of bioactive phytochemicals due to its high content in squalene.

Implications of farnesyltransferase and its inhibitors as a promising strategy for cancer therapy.

Ras proteins have been reported to play key role in oncologic diseases. Ras proteins are associated with cellular membranes for its carcinogenic activities through post-translational modifications, including farnesylation. Farnesyltransferase is responsible for a type of Ras membrane targeting, which leads to cancer origin and progression. Inhibitors of farnesyltransferase have been developed as novel anticancer agents. In this review, the role of farnesyltransferase in cancer progression and development has been discussed. Further, the current status of development of farnesyltransferase inhibitors for cancer prevention and treatment has also been reviewed.

Current insights into functions of phospholipase A2 receptor in normal and cancer cells: More questions than answers.

Lipid signaling network was proposed as a potential target for cancer prevention and treatment. Several recent studies revealed that phospholipid metabolising enzyme, phospholipase A2 (PLA2), is a critical regulator of cancer accelerating pathologies and apoptosis in several types of cancers. In addition to functioning as an enzyme, PLA2 can activate a phospholipase A2 receptor (PLA2R1) in plasma membrane. While the list of PLA2 targets extends to glucose homeostasis, intracellular energy balance, adipocyte development, and hepatic lipogenesis, the PLA2R1 downstream effectors are few and scarcely investigated. Among the most addressed PLA2R1 effects are regulation of pro-inflammatory signaling, autoimmunity, apoptosis, and senescence. Localized in glomeruli podocytes, the receptor can be identified by circulating anti-PLA2R1 autoantibodies leading to development of membranous nephropathy, a strong autoimmune inflammatory cascade. PLA2R1 was shown to induce activation of Janus-kinase 2 (JAK2) and estrogen-related receptor α (ERRα)-controlled mitochondrial proteins, as well as increasing the accumulation of reactive oxygen species, thus leading to apoptosis and senescence. These findings indicate the potential role of PLA2R1 as tumor suppressor. Epigenetic investigations addressed the role of DNA methylation, histone modifications, and specific microRNAs in the regulation of PLA2R1 expression. However, involvement of PLA2R1 in suppression of malignant growth and metastasis remains controversial. In this review, we summarize the recent findings that highlight the role of PLA2R1 in the regulation of carcinogenesis-related intracellular signaling.

Small molecule tyrosine kinase inhibitors and pancreatic cancer-Trials and troubles.

Pancreatic cancer (PC) is an aggressive carcinoma and the fourth cause of cancer deaths in Western countries. Although surgery is the most effective therapeutic option for PC, the management of unresectable, locally advanced disease is highly challenging. Our improved understanding of pancreatic tumor biology and associated pathways has led to the development of various treatment modalities that can control the metastatic spread of PC. This review intends to present trials of small molecule tyrosine kinase inhibitors (TKIs) in PC management and the troubles encountered due to inevitable acquired resistance to TKIs.

Ebola Therapeutic Study and Future Directions.

The constant Ebola epidemic outbreaks in Africa arisen in waves of panic worldwide. There is a high mortality rate (30-70%) among the Ebola-infected people in virus- stricken areas. Despite these horrors, the medical capabilities against this deadly viral disease were provided by limited therapeutic agents/options. As a result, several patented agents, biotherapies or prophylactic/therapeutic vaccines need to be reviving into the global markets-including patents of small molecular chemicals, short sequences or oligomers of DNA/RNA, linkages of chemicals with bio-molecules, herbal medicine and so on. In addition, the possible mechanisms of action of these therapeutic options are underway. To promote Ebola biomedical study, the multiple characters of Ebola infections-its origin, pathologic progress, genomic changes, therapeutic context and economic considerations are outlined in this review. Finally, a great difference can be expected after these types of efforts.

Role of GTPases in the Regulation of Mitochondrial Dynamics in Alzheimer's Disease and CNS-Related Disorders.

Data obtained from several studies have shown that mitochondria are involved and play a central role in the progression of several distinct pathological conditions. Morphological alterations and disruptions on the functionality of mitochondria may be related to metabolic and energy deficiency in neurons in a neurodegenerative disorder. Several recent studies demonstrate the linkage between neurodegeneration and mitochondrial dynamics in the spectrum of a promising era called precision mitochondrial medicine. In this review paper, an analysis of the correlation between mitochondria, Alzheimer's disease, and other central nervous system (CNS)-related disorders like the Parkinson's disease and the autism spectrum disorder is under discussion. The role of GTPases like the mfn1, mfn2, opa1, and dlp1 in mitochondrial fission and fusion is also under investigation, influencing mitochondrial population and leading to oxidative stress and neuronal damage.

Regulatory role of sphingosine kinase and sphingosine-1-phosphate receptor signaling in progenitor/stem cells.

Balanced sphingolipid signaling is important for the maintenance of homeostasis. Sphingolipids were demonstrated to function as structural components, second messengers, and regulators of cell growth and survival in normal and disease-affected tissues. Particularly, sphingosine kinase 1 (SphK1) and its product sphingosine-1-phosphate (S1P) operate as mediators and facilitators of proliferation-linked signaling. Unlimited proliferation (self-renewal) within the regulated environment is a hallmark of progenitor/stem cells that was recently associated with the S1P signaling network in vasculature, nervous, muscular, and immune systems. S1P was shown to regulate progenitor-related characteristics in normal and cancer stem cells (CSCs) via G-protein coupled receptors S1Pn (n = 1 to 5). The SphK/S1P axis is crucially involved in the regulation of embryonic development of vasculature and the nervous system, hematopoietic stem cell migration, regeneration of skeletal muscle, and development of multiple sclerosis. The ratio of the S1P receptor expression, localization, and specific S1P receptor-activated downstream effectors influenced the rate of self-renewal and should be further explored as regeneration-related targets. Considering malignant transformation, it is essential to control the level of self-renewal capacity. Proliferation of the progenitor cell should be synchronized with differentiation to provide healthy lifelong function of blood, immune systems, and replacement of damaged or dead cells. The differentiation-related role of SphK/S1P remains poorly assessed. A few pioneering investigations explored pharmacological tools that target sphingolipid signaling and can potentially confine and direct self-renewal towards normal differentiation. Further investigation is required to test the role of the SphK/S1P axis in regulation of self-renewal and differentiation.

Single Probiotic versus Multiple Probiotics - A Debate On Current Scenario for Alleviating Health Benefits.

Strain, dose, duration, validated clinical trial studies and scientific substantiation of health claims are needed in order to define a successful probiotic. In particular, the potency of probiotic differs from strain to strain. In this view, we aimed to know whether the single strain or multi-strain construct is more beneficial. We found that the studies were still under debate and mechanisms remain sometimes unclear. No conclusion can be made based on the strain experimentation. Safety data and significant statistical results for efficacy via well-controlled clinical trials are needed.