Capsicum chinense Jacq.-derived glutaredoxin (CcGRXS12) alters redox status of the cells to confer resistance against pepper mild mottle virus (PMMoV-I).

KEY MESSAGE: The CcGRXS12 gene protects plants from cellular oxidative damage that are caused by both biotic and abiotic stresses. The protein possesses GSH-disulphide oxidoreductase property but lacks Fe-S cluster assembly mechanism. Glutaredoxins (Grxs) are small, ubiquitous and multi-functional proteins. They are present in different compartments of plant cells. A chloroplast targeted Class I GRX (CcGRXS12) gene was isolated from Capsicum chinense during the pepper mild mottle virus (PMMoV) infection. Functional characterization of the gene was performed in Nicotiana benthamiana transgenic plants transformed with native C. chinense GRX (Nb:GRX), GRX-fused with GFP (Nb:GRX-GFP) and GRX-truncated for chloroplast sequences fused with GFP (Nb:Δ2MGRX-GFP). Overexpression of CcGRXS12 inhibited the PMMoV-I accumulation at the later stage of infection, accompanied with the activation of salicylic acid (SA) pathway pathogenesis-related (PR) transcripts and suppression of JA/ET pathway transcripts. Further, the reduced accumulation of auxin-induced Glutathione-S-Transferase (pCNT103) in CcGRXS12 overexpressing lines indicated that the protein could protect the plants from the oxidative stress caused by the virus. PMMoV-I infection increased the accumulation of pyridine nucleotides (PNs) mainly due to the reduced form of PNs (NAD(P)H), and it was high in Nb:GRX-GFP lines compared to other transgenic lines. Apart from biotic stress, CcGRXS12 protects the plants from abiotic stress conditions caused by H2O2 and herbicide paraquat. CcGRXS12 exhibited GSH-disulphide oxidoreductase activity in vitro; however, it was devoid of complementary Fe-S cluster assembly mechanism found in yeast. Overall, this study proves that CcGRXS12 plays a crucial role during biotic and abiotic stress in plants.

Expression and Immobilization of Tannase for Tannery Effluent Treatment from Lactobacillus plantarum and Staphylococcus lugdunensis: A Comparative Study.

Agro-industrial discharges have higher concentrations of tannins and have been a significant cause of pollution to water bodies and soil surrounding the agro-industries. So in this study, toxic tannic acid is into commercially valuable gallic acid from the tannery effluent using immobilized microbial tannase. Tannase genes were isolated from Lactobacillus plantarum JCM 1149 (tanLpl) and Staphylococcus lugdunensis MTCC 3614 (tanA). Further, these isolated tannese genes were cloned and expressed in BL 21 host using pET 28a as an expression vector,  and immobilized in sodium alginate beads. Vegetable tannery effluent was treated by tannase-immobilized beads at 25 °C and 37 °C, where liberated gallic acid was analyzed using TLC and NMR to confirm the tannin reduction. Further, both immobilized tannases exhibited excellent reusability up to 15 cycles of regeneration without significant reduction in their activity. Moreover, we also showed that immobilized tannases tanLpl and tanA activity remained unaffected compared to the free enzyme in the presence of metal ions. Further, tanA activity remained unaffected over a wide range of pH, and tanLpl showed high thermal stability. Thus, immobilized tannase tanLpl and tanA provide a possible solution for tannery effluent treatment depending upon industry requirements and reaction composition/effluent composition, one can choose a better-immobilized tannase among the two as per the need-based requirement.

Vitamin C fortification: need and recent trends in encapsulation technologies.

The multifaceted role of vitamin C in human health intrudes several biochemical functions that are but not limited to antioxidant activity, homoeostasis, amino acid synthesis, collagen synthesis, osteogenesis, neurotransmitter production and several yet to be explored functions. In absence of an innate biosynthetic pathway, humans are obligated to attain vitamin C from dietary sources to maintain its optimal serum level (28 µmol/L). However, a significant amount of naturally occurring vitamin C may deteriorate due to food processing, storage and distribution before reaching to the human gastrointestinal tract, thus limiting or mitigating its disease combating activity. Literature acknowledges the growing prevalence of vitamin C deficiency across the globe irrespective of geographic, economic and population variations. Several tools have been tested to address vitamin C deficiency, which are primarily diet diversification, biofortification, supplementation and food fortification. These strategies inherit their own advantages and limitations. Opportunely, nanotechnology promises an array of delivery systems providing encapsulation, protection and delivery of susceptible compounds against environmental factors. Lack of clear understanding of the suitability of the delivery system for vitamin C encapsulation and fortification; growing prevalence of its deficiency, it is a need of the hour to develop and design vitamin C fortified food ensuring homogeneous distribution, improved stability and enhanced bioavailability. This article is intended to review the importance of vitamin C in human health, its recommended daily allowance, its dietary sources, factors donating to its stability and degradation. The emphasis also given to review the strategies adopted to address vitamin c deficiency, delivery systems adopted for vitamin C encapsulation and fortification.

Recent Development and Application of "Nanozyme" Artificial Enzymes-A Review.

Nanozymes represent a category of nano-biomaterial artificial enzymes distinguished by their remarkable catalytic potency, stability, cost-effectiveness, biocompatibility, and degradability. These attributes position them as premier biomaterials with extensive applicability across medical, industrial, technological, and biological domains. Following the discovery of ferromagnetic nanoparticles with peroxidase-mimicking capabilities, extensive research endeavors have been dedicated to advancing nanozyme utilization. Their capacity to emulate the functions of natural enzymes has captivated researchers, prompting in-depth investigations into their attributes and potential applications. This exploration has yielded insights and innovations in various areas, including detection mechanisms, biosensing techniques, and device development. Nanozymes exhibit diverse compositions, sizes, and forms, resembling molecular entities such as proteins and tissue-based glucose. Their rapid impact on the body necessitates a comprehensive understanding of their intricate interplay. As each day witnesses the emergence of novel methodologies and technologies, the integration of nanozymes continues to surge, promising enhanced comprehension in the times ahead. This review centers on the expansive deployment and advancement of nanozyme materials, encompassing biomedical, biotechnological, and environmental contexts.

Assessment of metal contaminants along the Bay of Bengal - Multivariate pollution indices.

The spatial concentration of heavy metals (Mn, Ni, Cu, Co, Zn, Cd, and Pb) was studied in coastal areas (n = 9) including water (n = 27) and sediment (n = 27) in the Palk Bay, India to understand the metal pollution due to prevailing natural and anthropogenic activities. Pollution indices like metal index (MI), geoaccumulation index (Igeo), contamination factor (CF), pollution load index (PLI) and potential ecological risk (PER) were calculated based on the background/reference value. The values of MI index indicated that water was free of metals, whereas Igeo, CF, PLI and PER indicated moderate contamination of sediment in monsoon. Cadmium concentrations were the highest irrespective of the indices (Igeo: 0.04-1.42, Cf: 0.36-0.74, PLI: 0.36-0.74, and PER: 76.89-143.36) indicating moderate pollution. The Principal Component Analysis (PCA) affirmed that Cd was positively correlated with stations indicating anthropogenic sources of Cd contamination.

Lipid ROS- and Iron-Dependent Ferroptotic Cell Death in Unicellular Algae Chlamydomonas reinhardtii.

The phenomenon of heat stress leading to ferroptosis-like cell death has recently been observed in bacteria as well as plant cells. Despite recent findings, the evidence of ferroptosis, an iron-dependent cell death remains unknown in microalgae. The present study aimed to investigate if heat shock could induce reactive oxygen species (ROS) and iron-dependent ferroptotic cell death in Chlamydomonas reinhardtii in comparison with RSL3-induced ferroptosis. After RSL3 and heat shock (50 °C) treatments with or without inhibitors, Chlamydomonas cells were evaluated for cell viability and the induction of ferroptotic biomarkers. Both the heat shock and RSL3 treatment were found to trigger ferroptotic cell death, with hallmarks of glutathione-ascorbic acid depletion, GPX5 downregulation, mitochondrial dysfunction, an increase in cytosolic calcium, ROS production, lipid peroxidation, and intracellular iron accumulation via heme oxygenase-1 activation (HO-1). Interestingly, the cells preincubated with ferroptosis inhibitors (ferrostatin-1 and ciclopirox) significantly reduced RSL3- and heat-induced cell death by preventing the accumulation of Fe2+ and lipid ROS. These findings reveal that ferroptotic cell death affects the iron homeostasis and lipid peroxidation metabolism of Chlamydomonas, indicating that cell death pathways are evolutionarily conserved among eukaryotes.

Adsorption isotherm, kinetics and response surface methodology optimization of cadmium (Cd) removal from aqueous solution by chitosan biopolymers from cephalopod waste.

The present investigation was aimed to explore the cadmium removal efficiency, mechanism and characterization of Chitosan biopolymers from cephalopods waste. The extracted chitosan has showed good yield of 32% and with high minerals, ash and moisture content. In the Fourier-transform infrared spectroscopy (FT-IR) analysis multiple active functional groups of Amine, Amine, Hydroxyl were found between 612 and 3424 cm-1 and the sugar signals such as N-acetyl glucosamine (GlcNAc) and H-1 [GlcN (H-1D), GlcNAc (H-1A)] were identified in Chitosan by 1H Nuclear Magnetic Resonance (NMR). The Crystalline, rough surface, micropores characters were observed in Chitosan surface by Scanning Electron Microscope (SEM) analysis and the pores played a key role in adsorption process. The Cd ions removal was performed by batch experiment and the results were revealed that the pH, temperature, time and dosage highly influenced the process and the optimum condition was discovered through RSM for pH 7, temperature 42.5 °C, time 220 min and dosage of sorbent 1 g/L respectively. The kinetics models of the Cd removal were carried out and the results revealed that the Pseudo-second order is more suitable and fit for removal than Pseudo-first order model. Chitosan surface characters and functional groups played a big role in adsorption process and Chitosan can be alternative eco-friendly, low cost and highly efficient sorbent for heavy metal removal in effluent treatment plants.

Adaptive convergent evolution of genome proofreading in SARS-CoV2: insights into the Eigen's paradox

Evolutionary history of coronaviruses holds the key to understand mutational behavior and prepare for possible future outbreaks. By performing comparative genome analysis of nidovirales that contain the family of coronaviruses, we traced the origin of proofreading, surprisingly to the eukaryotic antiviral component ZNFX1. This common recent ancestor contributes two zinc finger (ZnF) motifs that are unique to viral exonuclease, segregating them from DNA proof-readers. Phylogenetic analyses indicate that following acquisition, genomes of coronaviruses retained and further fine-tuned proofreading exonuclease, whereas related families harbor substitution of key residues in ZnF1 motif concomitant to a reduction in their genome sizes. Structural modelling followed by simulation suggests the role of ZnF in RNA binding. Key ZnF residues strongly coevolve with replicase, and the helicase involved in duplex RNA unwinding. Hence, fidelity of replication in coronaviruses is a result of convergent evolution, that enables maintenance of genome stability akin to cellular proofreading systems.

Ectopic expression of bacterial 1-aminocyclopropane 1-carboxylate deaminase in Chlamydomonas reinhardtii enhances algal biomass and lipid content under nitrogen deficit condition.

1-Aminocyclopropane-1-carboxylate (ACC) deaminase is a well-known bacterial producing enzyme that helps plants to overcome stress conditions by modulating ethylene biosynthesis. However, the functional role of ACC deaminase and ethylene in microalgae during stress remains to be explored. In this study, to investigate the role of ACC deaminase (acds) from Pseudomonas putida UW4 in enhancing the biomass and lipid content of Chlamydomonas under nitrogen deficit condition. The synthetic codon-optimized acds gene was cloned into vector pChlamy_4 and introduced into Chlamydomonas. Results indicated that Chlamydomonas-expressing acds lines showed significant tolerance to nitrogen-deficit by reducing the ethylene content. The biomass, chlorophyll content and photosynthetic activity of acds-expressing lines were significantly increased during nitrogen deficit condition. Moreover, the intracellular lipid and fatty acid content were much higher in acds-expressing lines than the wild-type. In terms of stress alleviation, the transgenic lines displayed increased antioxidant enzymes, reduced ROS and lipid peroxidation levels.

Recent progress on engineering microbial alginate lyases towards their versatile role in biotechnological applications.

Biomass feedstock is an efficient and harmless source of energy. There are various sources of feedstock, such as plant, microbial, macro, and microalgae, and agricultural waste. The major component in biomass feedstock material is a polysaccharide, such as cellulose, cellobiose, starch, and alginate. Alginate is mainly found in macroalgae as one of the significant polysaccharide components. It is made up of ß-d-mannuronate (M) and α-l-guluronate (G) blocks. Alginate lyase is an enzyme that degrades alginate by breaking the glycosidic linkage between the poly M and G blocks to liberate oligosaccharides. Several organisms, including bacteria, fungi, viruses, and algae can produce alginate lyases. The species of bacteria, such as Bacillus, Vibrio, Pseudomonas, and Microbulbifer, are some of the important sources of alginate lyases. They are industrially essential enzymes used in food, biofuel, and biomedical industries. There are various assays available to determine the alginate lyase activity qualitatively as well as quantitatively. Qualitatively, different dyes like Gram's iodine, cetyl pyridinium chloride, and rutanium red can be used to visualize the zone formed due to the alginate lyase activity. DNS assay, UV absorption, and the Somogyi-Nelson method help to determine the alginate lyase activity quantitatively. Since the alginate lyase production in the native organisms is relatively lower, the genes encoding alginate lyases are heterologously cloned and expressed in E. coli to maximize the production and to characterize the enzyme. Different chromatographic techniques like size exclusion, affinity, gel permeation, and ion-exchange chromatography are used to purify the protein. In this paper, the source of alginate and alginate lyases, the mechanism of action of the enzyme, the engineering approaches to enhance the enzyme production, its purification strategy, and the potential applications of alginate lyases has been discussed.

Recombinant engineered phage-derived enzybiotic in Pichia pastoris X-33 as whole cell biocatalyst for effective biocontrol of Vibrio parahaemolyticus in aquaculture.

Vibrio parahaemolyticus is a major seafood-borne pathogen causing significant economic losses in aquaculture systems. Therefore, application of phage encoded enzymes, particularly endolysin, as a new strategy for effective biocontrol and therapeutic agent against bacterial diseases. In the present study, we synthesized endolysin gene (Vplys60) of bacteriophage qdv001 and biochemically characterized by expressing in Pichia pastoris X-33. In addition to, we also investigated the anti-biofilm and anti-vibriosis activity of Pichia-expressing Vplys60 against vibrio challenged in vivo aquaculture model, Artemia franciscana. The result indicated that the predicted molecular size of Pichia expressed Vplys60 was approximately 28 kDa as verified by SDS-PAGE and zymogram. Vplys60 manifested stable activity over broad range of pH (6-10), temperatures (37-75 °C) and salinity (100-600 mM NaCl). Biochemical and in silico analysis revealed that addition of calcium ion (Ca2+) enhanced the lytic activity of Vplys60 whereas other metal ions inhibited the activity. Additionally, calcium-dependent Vplys60 has showed a strong amidase activity by cleaving the peptidoglycan of V. parahaemolyticus. Our data also showed that Vplys60 (75 µg/ml) significantly inhibits biofilm formation (91.6%) and significantly reduced the bacterial population. The in vivo challenge study showed enhanced survival rate in combination with reduced vibrio load in Artemia after administration of Pichia-expressing Vplys60.

Bicarbonate supplementation enhances growth and biochemical composition of Dunaliella salina V-101 by reducing oxidative stress induced during macronutrient deficit conditions.

The unicellular marine alga Dunaliella salina is a most interesting green cell factory for the production of carotenes and lipids under extreme environment conditions. However, the culture conditions and their productivity are the major challenges faced by researchers which still need to be addressed. In this study, we investigated the effect of bicarbonate amendment on biomass, photosynthetic activity, biochemical constituents, nutrient uptake and antioxidant response of D. salina during macronutrient deficit conditions (N-, P- and S-). Under nutrient deficit conditions, addition of sodium bicarbonate (100 mM) significantly increased the biomass, carotenoids including ß-carotene and lutein, lipid, and fatty acid content with concurrent enhancement of the activities of nutrient assimilatory and carbonic anhydrase enzymes. Maximum accumulation of carotenoid especially ß-carotene (192.8 ± 2.11 µg/100 mg) and lipids (53.9%) was observed on addition of bicarbonate during nitrate deficiency compared to phosphate and sulphate deficiency. Supplementation of bicarbonate reduced the oxidative stress caused by ROS, lowered lipid peroxidation damage and improved the activities of antioxidant enzymes (SOD, CAT and APX) in D. salina cultures under nutrient stress.

Oral administration of lyophilized Dunaliella salina, a carotenoid-rich marine alga, reduces tumor progression in mammary cancer induced rats.

Dunaliella salina is a photosynthetic cell factory used for the commercial production of food additives, cattle stock feed and cosmetics as well as active ingredients for pharmaceutical industries. The investigation of the in vivo antitumor activity of D. salina lyophilized powder (DSLP) against 7,12-dimethylbenz(a)anthracene (DMBA) induced mammary carcinogenesis in female Wistar rats indicated a dose-dependent effect of DSLP. We studied the effect of DSLP at two different dosages of 500 and 1000 mg per kg bw on DMBA induced mammary cancer in rats by measuring the status of antioxidant enzymes, phase I and phase II detoxification enzymes, lipid peroxidation, and glycoconjugated proteins and by investigating the expression pattern of cell proliferation (Ki-67), hormonal receptor (ER, PR and HER2) status by immunohistochemical analysis, and apoptotic (caspase-3 and -9) and pro-inflammatory (COX-2) markers by colorimetric analysis. After 16 weeks of the study, we observed 100% tumor formation (including high tumor incidence and tumor volume) and a significant increase in the level of hormonal receptors, cell proliferation, and pro-inflammatory and apoptosis markers in tumor-bearing animals compared to the control. The oral administration of DSLP (1000 mg per kg bw) to the DMBA treated animals showed up to 83.4% reduction of tumors and effectively restored the levels of biochemical markers in the mammary tissues in addition to the downregulation of the expression of molecular markers. In conclusion, DSLP was found to show a chemopreventive effect against breast cancer induced in rats through the suppression of cell proliferation and the induction of apoptosis.

Accumulation of phytoene, a colorless carotenoid by inhibition of phytoene desaturase (PDS) gene in Dunaliella salina V-101.

The aim of this work was to study the accumulation of phytoene in Dunaliella salina V-101 by down-regulating its phytoene desaturase (PDS) gene expression using RNA interference and Antisense technology. RNAi and antisense constructs were introduced into the Dunaliella cells by Agrobacterium-mediated transformation. Among thirty-two transformants, six showed positive down-regulation of PDS expression with RNAi construct and five positive transformants were obtained using antisense construct. Characterization of PDS suppression was carried out using semi-quantitative RT-PCR and quantitative determination of phytoene as well as other carotenoids by HPLC. Both the RNAi and antisense lines showed a significant decrease in the expression levels of phytoene desaturase and carotenoid content compared to wild type cells. The RNAi line #5 showed maximum Phytoene content (108.34±22.34µg/100mg DCW) compared to other transgenic lines. These phytoene-accumulating phenotypes exhibited slower growth rates and were found to be sensitive to high light conditions.

Synergistic Action of D-Glucose and Acetosyringone on Agrobacterium Strains for Efficient Dunaliella Transformation.

An effective transformation protocol for Dunaliella, a ß-carotene producer, was developed using the synergistic mechanism of D-glucose and Acetosyringone on three different Agrobacterium strains (EHA105, GV3101 and LBA4404). In the present study, we investigated the pre-induction of Agrobacterium strains harboring pMDC45 binary vector in TAP media at varying concentrations of D-glucose (5 mM, 10 mM, and 15mM) and 100 µM of Acetosyringone for co-cultivation. Induction of Agrobacterium strains with 10 mM D-glucose and 100 µM Acetosyringone showed higher rates of efficiency compared to other treatments. The presence of GFP and HPT transgenes as a measure of transformation efficiency from the transgenic lines were determined using fluorescent microscopy, PCR, and southern blot analyzes. Highest transformation rate was obtained with the Agrobacterium strain LBA4404 (181 ± 3.78 cfu per 106 cells) followed by GV3101 (128 ± 5.29 cfu per 106 cells) and EHA105 (61 ± 5.03 cfu per 106 cells). However, the Agrobacterium strain GV3101 exhibited more efficient single copy transgene (HPT) transfer into the genome of D. salina than LBA4404. Therefore, future studies dealing with genetic modifications in D. salina can utilize GV3101 as an optimal Agrobacterium strain for gene transfer.

Engineering low phorbol ester Jatropha curcas seed by intercepting casbene biosynthesis.

KEY MESSAGE: Casbene is a precursor to phorbol esters and down-regulating casbene synthase effectively reduces phorbol ester biosynthesis. Seed-specific reduction of phorbol ester (PE) helps develop Jatropha seed cake for animal nutrition. Phorbol esters (PEs) are diterpenoids present in some Euphorbiaceae family members like Jatropha curcas L. (Jatropha), a tropical shrub yielding high-quality oil suitable as feedstock for biodiesel and bio jet fuel. Jatropha seed contains up to 40 % of oil and can produce oil together with cake containing high-quality proteins. However, skin-irritating and cancer-promoting PEs make Jatropha cake meal unsuitable for animal nutrition and also raise some safety and environmental concerns on its planting and processing. Two casbene synthase gene (JcCASA163 and JcCASD168) homologues were cloned from Jatropha genome and both genes were highly expressed during seed development. In vitro functional analysis proved casbene synthase activity of JcCASA163 in converting geranylgeranyl diphosphate into casbene which has been speculated to be the precursor to PEs. A seed-specific promoter driving inverted repeats for RNAi interference targeting at either JcCASA163 or both genes could effectively down-regulate casbene synthase gene expression with concurrent marked reduction of PE level (by as much as 85 %) in seeds with no pleiotropic effects observed. Such engineered low PE in seed was heritable and co-segregated with the transgene. Our work implicated casbene synthase in Jatropha PE biosynthesis and provided evidence for casbene being the precursor for PEs. The success in reducing seed PE content through down-regulation of casbene synthase demonstrates the feasibility of intercepting PE biosynthesis in Jatropha seed to help address safety concerns on Jatropha plantation and seed processing and facilitate use of its seed protein for animal nutrition.

Astaxanthin from psychrotrophic Sphingomonas faeni exhibits antagonism against food-spoilage bacteria at low temperatures.

Food production and processing industry holds a perpetual relationship with microorganisms and their by-products. In the present study, we aimed to identify beneficial cold-adapted bacteria devoid of any food spoilage properties and study their antagonism against common food-borne pathogens at low temperature conditions. Ten isolates were obtained on selective isolation at 5 °C, which were spread across genera Pseudomonas, Sphingomonas, Psychrobacter, Leuconostoc, Rhodococcus, and Arthrobacter. Methanol extracts of strains were found to contain several bioactive metabolites. Among the studied isolates, methanol extracts of S. faeni ISY and Rhodococcus fascians CS4 were found to show antagonism against growth of Escherichia coli, Proteus mirabilis, Enterobacter aerogenes, Listeria monocytogenes and Vibrio fischeri at refrigeration temperatures. Characterization of the abundant yellow pigment in methanol extracts of S. faeni ISY through UV-Vis spectrophotometry, high performance liquid chromatography (HPLC) and mass spectrometry (LC-MS) revealed the presence of astaxanthin, which, owing to its presence in very large amounts and evidenced to be responsible for antagonistic activity of the solvent extract.

Effect of dissolved inorganic carbon on ß-carotene and fatty acid production in Dunaliella sp.

This study aimed to explore the effect of sodium bicarbonate (0-200 mM) on the production of ß-carotene and lipid content in Dunaliella salina and Dunaliella bardawil. Total carotenoid and chlorophyll content were determined at regular intervals by a UV-VIS spectrophotometer. The ß-carotene and lipid contents were analyzed using high-performance liquid chromatography (HPLC) and gas chromatography coupled with mass spectrometry (GC-MS). The HPLC results revealed a twofold increase of ß-carotene in D. salina and D. bardawil cultures grown with sodium bicarbonate. Moreover, total fatty acid profiles from GC-MS indicated a maximum relative percentage of saturated fatty acids (tetradecanoic acid, 10,13-diethyl, methyl ester and methyl 16-methyl-heptadecanoate) compared to polyunsaturated fatty acids in both algae. Our results indicate that the optimum concentration of bicarbonate (100 to 150 mM) was required to stimulate a positive effect on ß-carotene production as well as the lipid profile in Dunaliella sp.

Metachronous multiple primary malignant neoplasms of the stomach and the breast: report of two cases with review of literature.

Multiple primary malignant neoplasm is the occurrence of a second primary malignancy in the same patient within 6 months of the detection of first primary (synchronous), or 6 months or more after primary detection (metachronous). Multiple primary malignant neoplasms are not very frequently encountered in clinical practice. The relative risk for a second primary malignancy increases by 1.111-fold every month from the detection of the first primary malignancy in any individual. We present 2 patients treated for carcinoma of the breast who developed a metachronous primary malignancy in the stomach to highlight the rare occurrence of multiple primary malignant neoplasms. These tumors were histologically dissimilar, with distinct immunohistochemical parameters. The importance lies in carefully identifying the second primary malignancies, not dismissing them as metastases, and treating them accordingly.