Topical Cream Carrying Drug-Loaded Nanogels for Melanoma Treatment.

In this study, nanogel creams carrying paclitaxel (PTX) and temozolomide (TMZ) were prepared for the topical treatment of melanoma. PTX and TMZ were first loaded in poly-(D,L-lactide-co-glycolide)-block-poly(ethylene glycol)-block-poly-(D,L-lactide-co-glycolide) (PLAG-b-PEG-b-PLGA) thermosensitive nanogels, which made a transition from a free-flowing sol (formation of micellar network) at 25°C with the z-average particle size of c.a. 96 nm to a gel (aggregation of micelles) at 33°C with the z-average particle size of c.a. 427 nm. An anhydrous absorption ointment base, aquaphor, was then added to drug-loaded nanogels to form nanogel creams carrying PTX and TMZ. Nanogel creams permitted controlled release of the payloads and improved the penetration of the payloads through the rodent skin compared to drug(s)-loaded nanogels. PTX and TMZ in a combination were synergistically effective in inhibiting SK-MEL28, A375, and B16-F10 melanoma cancer cells in vitro. Topically applied nanogel creams carrying TMZ/PTX (4 mg/1.5 mg/dose) showed a trend of tumor volume inhibition on B16-F10-bearing xenograft mice in vivo.

The Fatal Clinical Outcome of Severe COVID-19 in Hospitalized Patients: Findings from a Prospective Cohort Study in Dhaka, Bangladesh.

Background and Objectives: The morbidity and mortality associated with COVID-19 have burdened worldwide healthcare systems beyond their capacities, forcing them to promptly investigate the virus characteristics and its associated outcomes. This clinical analysis aimed to explore the key factors related to the fatal outcome of severe COVID-19 cases. Materials and Methods: Thirty-five adult severe COVID-19 patients were enrolled from two COVID-19 hospitals in Dhaka, Bangladesh. Clinical manifestation, comorbid conditions, medications, SARS-CoV-2 RT-PCR related cycle threshold (CT) value, hematology, biochemical parameters with SARS-CoV-2 specific IgG and IgM responses at enrollment were compared between the survivors and deceased participants. Results: Total 27 patients survived and 8 patients died within 3 months of disease onset. Deceased patients suffered longer from shortness of breath than the survived (p = 0.049). Among the severe cases, 62% of the deceased patients had multiple comorbid condition compared to 48% of those who survived. Interestingly, the anti-viral was initiated earlier among the deceased patients [median day of 1 (IQR: 0, 1.5) versus 6.5 (IQR: 6.25, 6.75)]. Most of the survivors (55%) received a combination of anticoagulant (p = 0.034). Liver enzymes, creatinine kinase, and procalcitonin were higher among the deceased patients during enrollment. The median CT value among the deceased was significantly lower than the survivors (p = 0.025). A significant difference for initial IgG (p = 0.013) and IgM (p = 0.030) responses was found between the survivor and the deceased groups. Conclusions: The factors including older age, male gender, early onset of respiratory distress, multiple comorbidities, low CT value, and poor antibody response may contribute to the fatal outcome in severe COVID-19 patients. Early initiation of anti-viral and a combination of anticoagulant treatment may prevent or lower the fatality among severe COVID-19 cases.

Extensive DNA damage-induced sumoylation contributes to replication and repair and acts in addition to the mec1 checkpoint.

The cellular response to DNA damage employs multiple dynamic protein modifications to exert rapid and adaptable effects. Substantial work has detailed the roles of canonical checkpoint-mediated phosphorylation in this program. Recent studies have also implicated sumoylation in the DNA damage response; however, a systematic view of the contribution of sumoylation to replication and repair and its interplay with checkpoints is lacking. Here, using a biochemical screen in yeast, we establish that DNA damage-induced sumoylation occurs on a large scale. We identify MRX (Mre11-Rad50-Xrs2) as a positive regulator of this induction for a subset of repair targets. In addition, we find that defective sumoylation results in failure to complete replication of a damaged genome and impaired DNA end processing, highlighting the importance of the SUMO-mediated response in genome integrity. We also show that DNA damage-induced sumoylation does not require Mec1 checkpoint signaling, and the presence of both enables optimal DNA damage resistance.

Small Molecules Targeting Biological Clock; A Novel Prospective for Anti-Cancer Drugs.

The circadian rhythms are an intrinsic timekeeping system that regulates numerous physiological, biochemical, and behavioral processes at intervals of approximately 24 h. By regulating such processes, the circadian rhythm allows organisms to anticipate and adapt to continuously changing environmental conditions. A growing body of evidence shows that disruptions to the circadian rhythm can lead to various disorders, including cancer. Recently, crucial knowledge has arisen regarding the essential features that underlie the overt circadian rhythm and its influence on physiological outputs. This knowledge suggests that specific small molecules can be utilized to control the circadian rhythm. It has been discovered that these small molecules can regulate circadian-clock-related disorders such as metabolic, cardiovascular, inflammatory, as well as cancer. This review examines the potential use of small molecules for developing new drugs, with emphasis placed on recent progress that has been made regarding the identification of small-molecule clock modulators and their potential use in treating cancer.

Circadian (De)regulation in Head and Neck Squamous Cell Carcinoma.

Head and neck cancer encompass different malignancies that develop in and around the throat, larynx, nose, sinuses and mouth. Most head and neck cancers are squamous cell carcinomas (HNSCC) that arise in the flat squamous cells that makeup the thin layer of tissue on the surface of anatomical structures in the head and neck. Each year, HNSCC is diagnosed in more than 600,000 people worldwide, with about 50,000 new cases. HNSCC is considered extremely curable if detected early. But the problem remains in treatment of inoperable cases, residues or late stages. Circadian rhythm regulation has a big role in developing various carcinomas, and head and neck tumors are no exception. A number of studies have reported that alteration in clock gene expression is associated with several cancers, including HNSCC. Analyses on circadian clock genes and their association with HNSCC have shown that expression of PER1, PER2, PER3, CRY1, CRY2, CKIε, TIM, and BMAL1 are deregulated in HNSCC tissues. This review paper comprehensively presents data on deregulation of circadian genes in HNSCC and critically evaluates their potential diagnostics and prognostics role in this type of pathology.

Conformational changes in a multidrug resistance ABC transporter DrrAB: Fluorescence-based approaches to study substrate binding.

Bacterial multidrug transporter DrrAB exhibits overlapping substrate specificity with mammalian P-glycoprotein. DrrA hydrolyzes ATP, and the energy is transduced to carrier DrrB resulting in export of drugs. Previous studies suggested that DrrB contains a large and flexible drug-binding pocket made of aromatic residues contributed by several transmembrane helices with different drugs binding to both specific and shared residues in this pocket. However, direct binding of drugs to DrrAB or the mechanism of substrate-induced conformational changes between DrrA and DrrB has so far not been investigated. We used two fluorescence-based approaches to determine substrate binding to purified DrrAB. Our analysis shows that DrrB binds drugs with variable affinities and contains multiple drug binding sites. This work also provides evidence for two asymmetric nucleotide binding sites in DrrA with strikingly different binding affinities. Using targeted fluorescence labeling, we provide clear evidence of long-range conformational changes occurring between DrrA and DrrB. It is proposed that the transduction pathway from the nucleotide-binding DrrA subunit to the substrate binding DrrB subunit includes Q-loop and CREEM motifs in DrrA and EAA-like motif in DrrB. This study lays a solid groundwork for examining roles of various conserved regions of DrrA and DrrB in transduction of conformational changes.

Cohesin recruits the Esco1 acetyltransferase genome wide to repress transcription and promote cohesion in somatic cells.

The cohesin complex links DNA molecules and plays key roles in the organization, expression, repair, and segregation of eukaryotic genomes. In vertebrates the Esco1 and Esco2 acetyltransferases both modify cohesin's Smc3 subunit to establish sister chromatid cohesion during S phase, but differ in their N-terminal domains and expression during development and across the cell cycle. Here we show that Esco1 and Esco2 also differ dramatically in their interaction with chromatin, as Esco1 is recruited by cohesin to over 11,000 sites, whereas Esco2 is infrequently enriched at REST/NRSF target genes. Esco1's colocalization with cohesin occurs throughout the cell cycle and depends on two short motifs (the A-box and B-box) present in and unique to all Esco1 orthologs. Deleting either motif led to the derepression of Esco1-proximal genes and functional uncoupling of cohesion from Smc3 acetylation. In contrast, other mutations that preserved Esco1's recruitment separated its roles in cohesion establishment and gene silencing. We conclude that Esco1 uses cohesin as both a substrate and a scaffold for coordinating multiple chromatin-based transactions in somatic cells.

Ubiquitin ligase Nedd4L targets activated Smad2/3 to limit TGF-beta signaling.

TGF-beta induces phosphorylation of the transcription factors Smad2 and Smad3 at the C terminus as well as at an interdomain linker region. TGF-beta-induced linker phosphorylation marks the activated Smad proteins for proteasome-mediated destruction. Here, we identify Nedd4L as the ubiquitin ligase responsible for this step. Through its WW domain, Nedd4L specifically recognizes a TGF-beta-induced phosphoThr-ProTyr motif in the linker region, resulting in Smad2/3 polyubiquitination and degradation. Nedd4L is not interchangeable with Smurf1, a ubiquitin ligase that targets BMP-activated, linker-phosphorylated Smad1. Nedd4L limits the half-life of TGF-beta-activated Smads and restricts the amplitude and duration of TGF-beta gene responses, and in mouse embryonic stem cells, it limits the induction of mesoendodermal fates by Smad2/3-activating factors. Hierarchical regulation is provided by SGK1, which phosphorylates Nedd4L to prevent binding of Smad2/3. Previously identified as a regulator of renal sodium channels, Nedd4L is shown here to play a broader role as a general modulator of Smad turnover during TGF-beta signal transduction.

Characterization of a novel domain 'GATE' in the ABC protein DrrA and its role in drug efflux by the DrrAB complex.

A novel domain, GATE (Glycine-loop And Transducer Element), is identified in the ABC protein DrrA. This domain shows sequence and structural conservation among close homologs of DrrA as well as distantly-related ABC proteins. Among the highly conserved residues in this domain are three glycines, G215, G221 and G231, of which G215 was found to be critical for stable expression of the DrrAB complex. Other conserved residues, including E201, G221, K227 and G231, were found to be critical for the catalytic and transport functions of the DrrAB transporter. Structural analysis of both the previously published crystal structure of the DrrA homolog MalK and the modeled structure of DrrA showed that G215 makes close contacts with residues in and around the Walker A motif, suggesting that these interactions may be critical for maintaining the integrity of the ATP binding pocket as well as the complex. It is also shown that G215A or K227R mutation diminishes some of the atomic interactions essential for ATP catalysis and overall transport function. Therefore, based on both the biochemical and structural analyses, it is proposed that the GATE domain, located outside of the previously identified ATP binding and hydrolysis motifs, is an additional element involved in ATP catalysis.

Cohesin acetylation speeds the replication fork.

Cohesin not only links sister chromatids but also inhibits the transcriptional machinery's interaction with and movement along chromatin. In contrast, replication forks must traverse such cohesin-associated obstructions to duplicate the entire genome in S phase. How this occurs is unknown. Through single-molecule analysis, we demonstrate that the replication factor C (RFC)-CTF18 clamp loader (RFC(CTF18)) controls the velocity, spacing and restart activity of replication forks in human cells and is required for robust acetylation of cohesin's SMC3 subunit and sister chromatid cohesion. Unexpectedly, we discovered that cohesin acetylation itself is a central determinant of fork processivity, as slow-moving replication forks were found in cells lacking the Eco1-related acetyltransferases ESCO1 or ESCO2 (refs 8-10) (including those derived from Roberts' syndrome patients, in whom ESCO2 is biallelically mutated) and in cells expressing a form of SMC3 that cannot be acetylated. This defect was a consequence of cohesin's hyperstable interaction with two regulatory cofactors, WAPL and PDS5A (refs 12, 13); removal of either cofactor allowed forks to progress rapidly without ESCO1, ESCO2, or RFC(CTF18). Our results show a novel mechanism for clamp-loader-dependent fork progression, mediated by the post-translational modification and structural remodelling of the cohesin ring. Loss of this regulatory mechanism leads to the spontaneous accrual of DNA damage and may contribute to the abnormalities of the Roberts' syndrome cohesinopathy.

Experimental study on tsunami risk reduction on coastal building fronted by sea wall.

This experimental study was conducted to idealize the efficacy of sea wall in controlling the tsunami forces on onshore structures. Different types of sea walls were placed in front of the building model. The tsunami forces and the wave heights were measured with and without the sea wall conditions. Types of sea wall, wall height, and wall positions were varied simultaneously to quantify the force reductions. Maximum of 41% forces was reduced by higher sea wall, positioned closer proximity to the model whereas this reduction was about 27% when the wall height was half of the high wall. Experimental investigations revealed that wall with adequate height and placed closer to the structures enables a satisfactory predictor of the force reduction on onshore structures. Another set of tests were performed with perforated wall placing near the building model. Less construction cost makes the provision of perforated sea wall interesting. The overall results showed that the efficacy of perforated wall is almost similar to solid wall. Hence, it can be efficiently used instead of solid wall. Moreover, overtopped water that is stuck behind the wall is readily gone back to the sea through perforations releasing additional forces on the nearby structures.

Sustainability of rainwater harvesting system in terms of water quality.

Water is considered an everlasting free source that can be acquired naturally. Demand for processed supply water is growing higher due to an increasing population. Sustainable use of water could maintain a balance between its demand and supply. Rainwater harvesting (RWH) is the most traditional and sustainable method, which could be easily used for potable and nonpotable purposes both in residential and commercial buildings. This could reduce the pressure on processed supply water which enhances the green living. This paper ensures the sustainability of this system through assessing several water-quality parameters of collected rainwater with respect to allowable limits. A number of parameters were included in the analysis: pH, fecal coliform, total coliform, total dissolved solids, turbidity, NH3-N, lead, BOD5, and so forth. The study reveals that the overall quality of water is quite satisfactory as per Bangladesh standards. RWH system offers sufficient amount of water and energy savings through lower consumption. Moreover, considering the cost for installation and maintenance expenses, the system is effective and economical.

Mechanistic insights from the atomic-level quaternary structure of short-lived GPCR oligomers in live cells.

The functional significance of the interactions between proteins in living cells to form short-lived quaternary structures cannot be overemphasized. Yet, quaternary structure information is not captured by current methods, neither can those methods determine structure within living cells. The dynamic versatility, abundance, and functional diversity of G protein-coupled receptors (GPCRs) pose myriad challenges to existing technologies but also present these proteins as the ideal testbed for new technologies to investigate the complex inter-regulation of receptor-ligand, receptor-receptor, and receptor-downstream effector interfaces in living cells. Here, we present development and use of a novel method capable of overcoming existing challenges by combining distributions (or spectrograms) of FRET efficiencies from populations of fluorescently tagged proteins associating into oligomeric complexes in live cells with diffusion-like trajectories of FRET donors and acceptors obtained from molecular dynamics (MD) simulations. Our approach provides an atom-level picture of the binding interfaces within oligomers of the human secretin receptor (hSecR) in live cells and allows for extraction of mechanistic insights into the function of GPCRs oligomerization. This FRET-MD spectrometry approach is a robust platform for investigating protein-protein binding mechanisms and opens a new avenue for investigating stable as well as fleeting quaternary structures of any membrane proteins in living cells.

Differential expression of HIV target cells CCR5 and α4ß7 in tissue resident memory CD4 T cells in endocervix during the menstrual cycle of HIV seronegative women.

Background: Ovarian hormones are known to modulate the immune system in the female genital tract (FGT). We sought to define the impact of the menstrual cycle on the mucosal HIV target cell levels, and tissue-resident CD4 T cells. Materials and methods: Here, we characterized the distribution, phenotype, and function of CD4 T cells with special emphasis on HIV target cells (CCR5+ and α4ß7+) as well as tissue-resident memory (TRM; CD69+ and CD103+) CD4 T cells in FGT of cycling women. Peripheral blood and Endocervical cells (EC-collected from cytobrush) were collected from 105 healthy women and performed multicolor flow cytometry to characterize the various subsets of CD4 T cells. Cervicovaginal lavage (CVL) were collected for cytokine analysis and plasma were collected for hormonal analysis. All parameters were compared between follicular and luteal phase of menstrual cycle. Results: Our findings revealed no significant difference in the blood CD4 T cell subsets between the follicular and luteal phase. However, in EC, the proportion of several cell types was higher in the follicular phase compared to the luteal phase of menstrual cycle, including CCR5+α4ß7-cells (p=0.01), CD69+CD103+ TRM (p=0.02), CCR5+CD69+CD103+ TRM (p=0.001) and FoxP3+ CD4 T cells (p=0.0005). In contrast, α4ß7+ CCR5- cells were higher in the luteal phase (p=0.0004) compared to the follicular phase. In addition, we also found that hormonal levels (P4/E2 ratio) and cytokines (IL-5 and IL-6) were correlated with CCR5+ CD4 T cells subsets during the follicular phase of the menstrual cycle. Conclusion: Overall, these findings suggest the difference in the expression of CCR5 and α4ß7 in TRM CD4 T cell subsets in endocervix of HIV seronegative women between the follicular and luteal phase. Increase in the CCR5+ expression on TRM subsets could increase susceptibility to HIV infection during follicular phase of the menstrual cycle.

An Integrated Approach Combining Experimental, Informatics and Energetic Methods for Solid Form Derisking of PF-06282999.

The landscapes of observed and predicted three-dimensional crystal packing arrangements of small-molecule drug candidates can be complex. The possible appearance of a more thermodynamically stable solid form during drug development has led to the digital workflow of informatics-based risk assessments, named a Solid Form Health Check. Herein, we describe the use of a combined approach consisting of experiments, informatics together with energetic calculations in analysis of four competing polymorphs of PF-06282999, a myeloperoxidase (MPO) inhibitor with conformational flexibility and multiple plausible hydrogen bond networks. This combined approach offered a comprehensive understanding of the solid form structure, properties, and performance, ensuring robust solid form derisking and selection.

A snap-shot of a diarrheal epidemic in Dhaka due to enterotoxigenic Escherichia coli and Vibrio cholerae O1 in 2022.

Background: Enterotoxigenic Escherichia coli (ETEC) and Vibrio cholerae O1 are most common bacterial causes of diarrheal diseases in Bangladesh. This analysis projected distribution of ETEC and V. cholerae O1 among diarrheal patients of icddr,b, Dhaka hospital in two diarrheal peaks of 2022. Methodology: Under the 2% systematic surveillance system, stool samples collected from diarrheal patients of icddr,b hospital were cultured and diagnostic testing was done for ETEC and V. cholerae O1. Comparison of positive cases was done between first peak (March-April) and second peak (October-November) in 2022. Results: A total of 2,937 stool specimens were tested of which 12% were ETEC and 20% were V. cholerae O1. About 40% of the severe dehydration cases were infected with V. cholerae O1. Predominant ETEC enterotoxin type was 'LT/ST' (41%). The LT enterotoxin significantly increased from 13% to 28% in the second peak (p = 0.015). The predominant colonization factors (CFs) on ETEC were CS5 + CS6 (23%), followed by CS6 (15%). CF-positive isolates was significantly higher in the second peak (36%) than in the first peak (22%) (p = 0.043). Total 14% cases were co-infected with ETEC and V. cholerae O1. Significant differences in the distribution of enterotoxin types were observed (p = 0.029) among the co-infection cases. Conclusion: Changing patterns of enterotoxin and CFs observed in ETEC pathogens should be taken into consideration for ETEC vaccine development. Considering cholera and ETEC biannual trends in causing diarrheal epidemics and outbreaks, emphasizes the need for thoughts on combination vaccine strategies for preventing acute watery diarrhea due to the two major bacterial pathogens.

Determining clinical biomarkers to predict long-term SARS-CoV-2 antibody response among COVID-19 patients in Bangladesh.

Background: Information on antibody responses following SARS-CoV-2 infection, including the magnitude and duration of responses, is limited. In this analysis, we aimed to identify clinical biomarkers that can predict long-term antibody responses following natural SARS-CoV-2 infection. Methodology: In this prospective study, we enrolled 100 COVID-19 patients between November 2020 and February 2021 and followed them for 6 months. The association of clinical laboratory parameters on enrollment, including lactate dehydrogenase (LDH), neutrophil-lymphocyte ratio (NLR), C-reactive protein (CRP), ferritin, procalcitonin (PCT), and D-dimer, with predicting the geometric mean (GM) concentration of SARS-CoV-2 receptor-binding domain (RBD)-specific IgG antibody at 3 and 6 months post-infection was assessed in multivariable linear regression models. Result: The mean ± SD age of patients in the cohort was 46.8 ± 14 years, and 58.8% were male. Data from 68 patients at 3 months follow-up and 55 patients at 6 months follow-up were analyzed. Over 90% of patients were seropositive against RBD-specific IgG till 6 months post-infection. At 3 months, for any 10% increase in absolute lymphocyte count and NLR, there was a 6.28% (95% CI: 9.68, -2.77) decrease and 4.93% (95% CI: 2.43, 7.50) increase, respectively, in GM of IgG concentration, while any 10% increase for LDH, CRP, ferritin, and procalcitonin was associated with a 10.63, 2.87, 2.54, and 3.11% increase in the GM of IgG concentration, respectively. Any 10% increase in LDH, CRP, and ferritin was similarly associated with an 11.28, 2.48, and 3.0% increase in GM of IgG concentration at 6 months post-infection. Conclusion: Several clinical biomarkers in the acute phase of SARS-CoV-2 infection are associated with enhanced IgG antibody response detected after 6 months of disease onset. The measurement of SARS-CoV-2 specific antibody responses requires improved techniques and is not feasible in all settings. Baseline clinical biomarkers can be a useful alternative as they can predict antibody response during the convalescence period. Individuals with an increased level of NLR, CRP, LDH, ferritin, and procalcitonin may benefit from the boosting effect of vaccines. Further analyses will determine whether biochemical parameters can predict RBD-specific IgG antibody responses at later time points and the association of neutralizing antibody responses.

Appearance of tolerance-induction and non-inflammatory SARS-CoV-2 spike-specific IgG4 antibodies after COVID-19 booster vaccinations.

Background: Understanding the characteristics of the humoral immune responses following COVID-19 vaccinations is crucial for refining vaccination strategies and predicting immune responses to emerging SARS-CoV-2 variants. Methods: A longitudinal analysis of SARS-CoV-2 spike receptor binding domain (RBD) specific IgG antibody responses, encompassing IgG subclasses IgG1, IgG2, IgG3, and IgG4 was performed. Participants received four mRNA vaccine doses (group 1; n=10) or two ChAdOx1 nCoV-19 and two mRNA booster doses (group 2; n=19) in Bangladesh over two years. Results: Findings demonstrate robust IgG responses after primary Covishield or mRNA doses; declining to baseline within six months. First mRNA booster restored and surpassed primary IgG responses but waned after six months. Surprisingly, a second mRNA booster did not increase IgG levels further. Comprehensive IgG subclass analysis showed primary Covishield/mRNA vaccination generated predominantly IgG1 responses with limited IgG2/IgG3, Remarkably, IgG4 responses exhibited a distinct pattern. IgG4 remained undetectable initially but increased extensively six months after the second mRNA dose, eventually replacing IgG1 after the 3rd/4th mRNA doses. Conversely, initial Covishield recipients lack IgG4, surged post-second mRNA booster. Notably, mRNA-vaccinated individuals displayed earlier, robust IgG4 levels post first mRNA booster versus Covishield counterparts. IgG1 to IgG4 ratios decreased with increasing doses, most pronounced with four mRNA doses. This study highlights IgG response kinetics, influenced by vaccine type and doses, impacting immunological tolerance and IgG4 induction, shaping future vaccination strategies. Conclusions: This study highlights the dynamics of IgG responses dependent on vaccine type and number of doses, leading to immunological tolerance and IgG4 induction, and shaping future vaccination strategies.

Multicentric Castleman Disease: The Hyaline Vascular Type Without HIV Association in a Young Male With Cutaneous Manifestation and Renal Impairment.

Castleman disease (CD), a heterogenous lymphoproliferative disorder resulting from immune dysregulation, is a very rare disease in clinical practice. The clinical spectrum of Castleman disease is wide and its treatment options are mostly based on case reports and case series. To date, two clinical and four histological types have been described. It has recently been successfully demonstrated that the pathogenesis of multicentric Castleman disease (MCD) has a direct association with human immunodeficiency virus (HIV) and human herpes virus 8 (HHV-8) infection which is why further studies are necessary. Here, we report an unusual case of MCD not associated with HIV and having a histological diagnosis of the hyaline vascular type that presented with acute renal impairment and subcutaneous abnormal lymphatic proliferation.

Fluorescence Intensity Fluctuation Analysis of Protein Oligomerization in Cell Membranes.

Fluorescence fluctuation spectroscopy (FFS) encompasses a bevy of techniques that involve analyzing fluorescence intensity fluctuations occurring due to fluorescently labeled molecules diffusing in and out of a microscope's focal region. Statistical analysis of these fluctuations may reveal the oligomerization (i.e., association) state of said molecules. We have recently developed a new FFS-based method, termed Two-Dimensional Fluorescence Intensity Fluctuation (2D FIF) spectrometry, which provides quantitative information on the size and stability of protein oligomers as a function of receptor concentration. This article describes protocols for employing FIF spectrometry to quantify the oligomerization of a membrane protein of interest, with specific instructions regarding cell preparation, image acquisition, and analysis of images given in detail. Application of the FIF Spectrometry Suite, a software package designed for applying FIF analysis on fluorescence images, is emphasized in the protocol. Also discussed in detail is the identification, removal, and/or analysis of inhomogeneous regions of the membrane that appear as bright spots. The 2D FIF approach is particularly suited to assess the effects of agonists and antagonists on the oligomeric size of membrane receptors of interest. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of live cells expressing protein constructs Basic Protocol 2: Image acquisition and noise correction Basic Protocol 3: Drawing and segmenting regions of interest Basic Protocol 4: Calculating the molecular brightness and concentration of individual image segments Basic Protocol 5: Combining data subsets using a manual procedure (Optional) Alternate Protocol 1: Combining data subsets using the advanced FIF spectrometry suite (Optional; alternative to Basic Protocol 5) Basic Protocol 6: Performing meta-analysis of brightness spectrograms Alternate Protocol 2: Performing meta-analysis of brightness spectrograms (alternative to Basic Protocol 6) Basic Protocol 7: Spot extraction and analysis using a manual procedure or by writing a program (Optional) Alternate Protocol 3: Automated spot extraction and analysis (Optional; alternative to Protocol 7) Support Protocol: Monomeric brightness determination.