Issue Date: July 2022
Common Milk Protein Blocks COVID-19 Infections in Cultured Human Cells
- Researchers are trying to understand whether dairy plays a protective role against COVID-19 infection.
- The milk protein lactoferrin prevents SARS-CoV2 from infecting cells in lab experiments.
- Future studies could yield a potential antiviral pill that reduces viral transmission.
Early during the COVID-19 pandemic, medicinal chemist Jonathan Sexton of the University of Michigan and his colleagues were screening a range of drugs to see if any might work as effective antivirals against the SARS-CoV2 virus. While setting up the experiment, Jesse Wotring, a graduate student in the lab, added one more molecule on an impulse: an iron-binding protein named lactoferrin found in many secretions, including bovine and human milk.
The researchers had lactoferrin on hand because they were studying its potential benefits in a different condition known as hemochromatosis, where iron accumulates in tissues and causes poisoning. But a few previous reports had suggested the molecule also had antiviral activity against common pathogens such as norovirus and could block SARS-1 by preventing the virus from binding to cell surface receptors known as heparan sulfate proteoglycans.
In their experiments, Sexton and his team tested a library of more than 1400 FDA-approved drugs in cultured human cells for their effects on SARS-CoV2 infection. They identified 17 promising candidates—and lactoferrin ranked especially high on the list, blocking viral entry into cells, and thus preventing infection . “It was hands down the most potent and the most effective therapy that we found,” Sexton says. “It was very exciting because of its wide availability as a dietary supplement.”
The researchers set out to test their serendipitous discovery and understand the underlying mechanisms that might be at work. To do so, they analyzed the transcriptome of cell lines that were treated with lactoferrin. They found that the treatment stimulated a strong interferon response that shut down viral replication inside cells. It also suppressed the production of inflammatory molecules that drive a runaway immune response known as the cytokine storm, which has been linked to severe outcomes of COVID-19 . “It seems to have three separate mechanisms for efficacy against SARS-CoV2,” Sexton says.
Lactoferrin is present in both human and bovine milk, and a handful of studies hint at dairy consumption playing a potential protective role in COVID-19 infections. But others have found the opposite effect, possibly because of the diverse kinds of dairy products studied by different researchers. In one recent example, Mahdieh Hosseinzadeh of the Shahid Sadoughi University of Medical Sciences in Iran and colleagues surveyed 8801 adults about their dairy consumption habits and correlated the information with the severity of their COVID-19 infections. Of the group surveyed, 474 had experienced COVID-19 infections. Their analysis found that a moderate intake of dairy products—especially low-fat products—correlated with a protective role against COVID-19 infections. People who consumed larger amounts of high-fat dairy products, including whole milk, cheese, and butter appeared to have a greater risk of COVID-19 infections . “Low-fat dairy products have more lactoferrin in them, so it’s interesting that their intake has a stronger protective role,” Sexton says.
But Sexton and the study authors add that while the results seem robust, they do not establish a cause-and-effect relationship between risk of COVID-19 infection and consuming dairy products. In their study, the authors write that the results, albeit promising, are “not enough to provide reliable nutritional recommendations, and stronger clinical studies are needed.”
In a follow-up to their 2021 study, Sexton and his team compared several lactoferrin-rich dairy products and supplements for their protective effects. They cultured human cells overnight, then treated them with a lactoferrin supplement, whey protein, and other products for 24 hours before infecting the cells with five variants of SARS-CoV2. Two days after infection, the team analyzed the cells using microscopy. They also tested a related iron-binding protein named transferrin and found no effects, confirming that lactoferrin’s antiviral effect is not dependent on the presence of iron alone . “The efficacy we saw with the whey protein isolates basically tracked with the amount of lactoferrin in the isolates, so it seems it is the main active ingredient,” Sexton says. “Some other milk peptides have some antiviral activity, but it’s really the lactoferrin that’s doing all of the heavy lifting in the context of SARS-CoV2 infection.”
The team is now testing an oral formulation of lactoferrin to increase its presence in the mouth and mucosal membranes of the upper gastro-intestinal tract. If it works, this could act as another way to stop viral spread, Sexton says. “The vaccines are highly effective at preventing severe disease, but they don’t give you mucosal immunity—even after being vaccinated, you can still contract and spread the virus,” he adds.
A version of lactoferrin that persists on mucosal membranes and blocks the virus from entering cells could help prevent transmission. It might be “particularly useful for prophylaxis,” Sexton says. “We need every tool we can get to end this pandemic.”
- Mirabelli C, Wotring JW, Zhang CJ, McCarty SM, Fursmidt R, Pretto CD, Qiao Y, Zhang Y, Frum T, Kadambi NS, Amin AT. Morphological cell profiling of SARS-CoV-2 infection identifies drug repurposing candidates for COVID-19. Proceedings of the National Academy of Sciences. 2021 Sep 7;118(36):e2105815118.
- Darand M, Hassanizadeh S, Marzban A, Mirzaei M, Hosseinzadeh M. The association between dairy products and the risk of COVID-19. European Journal of Clinical Nutrition. 2022 Apr 29:1-7.
- Wotring JW, Fursmidt R, Ward L, Sexton JZ. Evaluating the in vitro efficacy of bovine lactoferrin products against SARS-CoV-2 variants of concern. Journal of Dairy Science. 2022 Apr 1;105(4):2791-802.
First Steps towards Intranasal Milk Therapy for Preterm Infants
- Intraventricular hemorrhaging occurs in preterm infants when the delicate blood vessels in the brain rupture.
- This condition can to lead to neurodevelopmental problems, and there is currently no effective treatment.
- Human milk is full of stem cells, immune cells, and growth factors, which could have a protective effect against brain damage.
- Scientists proved the safety and feasibility of milk therapy by giving 37 preterm infants their mother’s milk intranasally for 28 days without adverse outcomes.
At the NICU at the Hospital for Sick Children in Toronto, Canada, Dr. Rebecca Hoban spends long days treating preterm infants born between 25 and 36 weeks at gestation. A common complication among her tiny patients is intraventricular hemorrhaging, a complication that occurs when the underdeveloped and fragile blood vessels in the brain rupture, and blood pools into the fluid-filled ventricles surrounding the brain . There is no effective treatment for intraventricular hemorrhaging, and neonatologists like Hoban are interested in exploring a potential new therapy using human milk given through the nose. In a first prospective trial, Hoban and her colleagues tested the safety and feasibility of such a therapy for preterm infants and presented their findings at the Pediatric Academic Societies meeting held in Denver, Colorado, earlier this year .
“Human milk has stem cells and other bioactives,” says Hoban. “That’s why we think it’s so helpful.” By administering milk through the nose, Hoban hopes these stem cells and other neuroprotectants could “potentially reduce inflammation and damage” through a more direct route to the brain. Down the line, intraventricular hemorrhaging can lead to neurodevelopmental problems such as hydroencephalus or cysts in the brain called porencephaly .
Previous studies have shown that human milk fed to preterm infants in the NICU helped with long-term neurological outcomes . However, the milk is typically fed using a gavage tube, a tube that runs from the nose to the stomach and bypasses the nasal passage to the brain . In healthy breastfeeding infants, the mother’s milk often gets into the infant’s nasal cavity . For this study, scientists were interested in the more direct route from the nose to the brain. Past studies conducted in neonatal mice have found that growth factors from milk helped promote recovery after brain damage .
Furthermore, a previous study retroactively examined 31 cases of preterm infants with severe intraventricular hemorrhaging, 16 of which were given nasal drops of human milk. Although the study results were not statistically significant, infants who received intranasal breast milk trended toward lower incidences of severe brain defects. The authors noted that future studies should be conducted with scientific controls, and highlighted the need for research into intranasal milk treatments. Very little is currently known about the mechanism behind how milk might attenuate brain damage in neonates .
In the newly presented research, Hoban and her colleague’s goal was to prove the safety and feasibility of giving intranasal milk, and to help develop a framework for future research that looks at the efficacy and long-term effects of the treatment .
The study was conducted on 37 infants who were under 33-weeks gestation and diagnosed with intraventricular hemorrhaging. Hoban and her colleagues used a small, oral syringe to carefully give infants doses of 0.2 mL of their own mother’s breast milk administered through the nose. If tolerated, the dosage was increased to 0.4 mL to a goal of 1.6 mL a day. The study took note of treatment tolerance, adverse effects, and other health measures .
“Our prospective trial in Toronto, the first in the world, showed that these preterm infants were able to tolerate nasal milk therapy through 28 days of life without major safety events,” says Alessia Gallipoli, one of the study co-authors.
One of the challenges, Hoban says, was figuring out the logistics of giving the infants their mother’s milk within 2–3 hours of lactation, as the parents were not always present or ready to provide milk. Given the nature of the study, finding enough study participants was also a lengthy process.
Future analyses will look at the efficacy of intranasal milk therapy and assess the longer-term effects to see if incidences of severe brain damage is reduced .
- Stanford Children’s Health. Intraventricular Hemorrhaging in Babies [internet]. Available from: https://www.stanfordchildrens.org/en/topic/default?id=intraventricular-hemorrhage-90-P02608
- Gallipoli A. (2022, April). Intranasal human milk as as stem cell therapy in preterm infants with intraventricular hemorrhage: safety, feasibility, and short term outcomes. Poster session presented at the meeting of Pediatric Academic Societies, Denver, Colorado.
- Vohr BR, Poindexter BB, Dusick AM, McKinley LT, Higgins RD, Langer JC. Persistent beneficial effects of breast milk ingested in the neonatal intensive care unit on outcomes of extremely low birth weight infants at 30 months of age. Pediatrics. 2007; 120: e953–e959
- Keller T, Körber F, Oberthuer A. Schafmeyer L, Mehler K, Kuhr K, Kribs A. Intranasal breast milk for premature infants with severe intraventricular hemorrhage—an observation. Eur J Pediatr. 2019;178:199–206
- van de Looij Y, Ginet V, Chatagner A, Toulotte A, Somm E, Hüppi PS, Sizonenko SV. Lactoferrin during lactation protects the immature hypoxic-ischemic rat brain. Ann Clin Transl Neurol. 2014; 1: 955–967
Fermented Dairy Foods Show Potential to Delay Cognitive Decline and Dementia
- Consumption of fermented dairy foods, including yogurt, kefir, and some types of cheese, has been associated with lower risks of many chronic diseases.
- Fermented dairy foods can positively alter the composition of the gut microbiome, which influences physiological, nutritional, metabolic, and immunological processes in the body.
- New research suggests that fermented dairy foods offer neuroprotective effects and could be used to treat and even prevent the progression of cognitive decline seen in dementia and Alzheimer’s disease.
Humans have been consuming fermented dairy foods for nearly 10,000 years, but it’s only recently these foods have attracted scientific attention for their potential health benefits. Fermenting milk prevents colonization and contamination by dangerous microorganisms, with a bonus of making dairy easier to digest by reducing the lactose content. But the process also enriches the final food product with beneficial bacteria, fungi, and biologically active peptides, ingredients that have anti-inflammatory and antioxidant effects on many aspects of human physiology [1, 2]. New research [2-6] extends these beneficial actions to the brain and suggests that consumption of fermented dairy foods could help slow the progression of dementia, including dementia caused by Alzheimer’s disease (AD). Could a prehistoric solution for milk preservation provide a modern-day solution for cognitive decline?
Food with a Function
Fermentation transforms the starter food in taste, texture, and aroma while imbuing it with health benefits not attributable to the starter food. This process sounds like something out of a Harry Potter spell book, but the “magic” is simply the result of microorganisms (bacteria, fungi, or both) breaking down carbohydrates from the starter food into simpler sugars, like alcohol, and organic acids. In yogurt, for example, lactic acid bacteria (LAB) break down lactose into (you guessed it!) lactic acid. These simpler sugars and acids act as natural preservatives by preventing the growth of dangerous microorganisms and give fermented foods their distinctive sour or tart taste .
Microorganisms used to ferment dairy foods also make enzymes that break apart milk proteins into smaller peptide chains and free amino acids. Some types of microorganisms, including LAB, even synthesize thiamin, riboflavin, and niacin, and can increase the bioavailability of certain minerals like calcium . These enzymatic activities are the reason that yogurt, kefir, and cheese look and taste different than milk and have nutritional and biological properties beyond those provided by milk, including live cultures of the very microorganisms that started the fermentation process.
These additional properties of fermented dairy foods make them functional foods, meaning they provide positive effects on human health beyond nutrients. Fermented dairy foods deliver microorganisms that are identical to or related to species that are well known for their probiotic activity (LAB, again!). The live cultures in yogurt and kefir can interact with microbes in the host’s gut, outcompete pathogenic strains, and improve the balance of healthy to unhealthy gut microbes [1-7]. Not surprisingly, this directly impacts the health of the intestines and improves digestion and nutrient absorption.
What may be a surprise is the number of physiological, metabolic, and immunological processes throughout the body that are impacted by a shift to a healthier gut microbiome . In the last decade, researchers have found that yogurt intake was associated with a lower risk for type 2 diabetes, cardiovascular disease, bladder cancer, chronic inflammation, and overall mortality, and both yogurt and kefir consumption have been linked to improvements in mental health.
This is Your Brain on Fermented Dairy
The results of these studies imply an association between chronic disease and gut dysbiosis (an imbalance in gut microbiota)  and sparked a considerable amount of research on the gut microbiomes of individuals suffering from other chronic diseases, including AD. Compared with healthy controls, gut microbiomes of mouse models of AD and human patients diagnosed with AD were distinct, with less diversity of bacterial species and a higher percentage of strains considered pro-inflammatory [9, 10]. In fact, the differences between healthy and AD gut microbiomes were so distinct that microbiome composition is being proposed as a diagnostic tool for AD .
Modifying the gut microbiome with fermented dairy foods offers a potential pathway for treating AD. The gut microbiome and the brain have a bi-directional mode of communication (called the gut-brain axis) via the central nervous system and the gut’s enteric nervous system, which has been called the “second brain” . Signals are sent between the two “brains” through chemical messengers such as hormones, cytokines, immune cells, and neurotransmitters. For example, certain strains of gut bacteria (looking at you, LAB) synthesize gamma-aminobutyric acid (GABA), a neurotransmitter that produces a calming effect. In humans and mice, GABA production from kefir consumption has been linked to improvements in mood.
Research on human subjects indicates consumption of fermented dairy foods brought about improvements in cognitive function, presumably through alterations in the gut microbiome. In a 12-week, double-blinded randomized control trial (RCT), a daily dose (200 ml/day or close to 1 cup) of a fermented milk product improved cognitive function in AD patients between 60 and 95 years of age [reviewed in 6]. Similar results were found with supplementation of healthy adults over 60 [reviewed in 6]. One RCT supplemented healthy older adults for 12 weeks with 180 grams per day (about 0.6 of a cup) of Lactobacillus helveticus (a strain of LAB) fermented in skim milk powder and found improved scores on cognitive tests and another used the same supplement for only eight weeks and noted significant improvement in attention and delayed memory in supplemented participants compared with controls [reviewed in 6].
These findings are remarkable for several reasons. First, the length of time the supplements were provided was between two and three months, and yet the investigators were able to demonstrate a statically significant effect of the supplement. This suggests that changes to the gut microbiome that elicit neuroprotective effects (e.g., anti-inflammatory or antioxidant) can happen rather rapidly. Second, the studies supplemented the participants with quantities similar to (or even less than) what one might consume daily, indicating drastic dietary modifications are not necessary. And finally, the studies were able to demonstrate an effect of diet on cognitive function in healthy patients, suggesting the potential to use fermented dairy foods in prevention of cognitive decline, not just in alleviating symptoms.
To understand what was happening at the cellular level to account for improved cognitive function in humans, researchers turned to mouse models of AD. AD is characterized by loss of neural synapses and neuronal damage caused by the presence of amyloid beta (Aβ) protein plaques around brain cells and intracellular neurofibrillary tangles (NFTs) made of tau protein within brain cells [reviewed in 4, 6]. Microglia are immune cells that scavenge and consume toxic cells in the central nervous system, playing an important role in clearing Aβ and regulating inflammation in the brain. In a mouse model for AD, camembert cheese fermented with the fungus Penicillium candidum reduced the build-up of Aβ protein plaques, decreased the production of inflammatory cytokines in the hippocampus, and improved microglial phagocytosis of Aβ plaque [reviewed in 4]. In another experiment, bioactive peptides produced from whey protein (WY peptides) during digestion of fermented dairy foods improved memory performance in mice by raising the levels of dopamine and monoamine (types of neurotransmitters) in brain tissue [reviewed in 4, 6].
The results of these and many other studies on fermented dairy foods and AD suggest that the benefits of fermented dairy consumption are not simply a generalized improvement in gut microbiome composition—microorganisms and their metabolites from fermented dairy foods elicit specific neuroprotective effects [2-6].
With current AD and dementia pharmaceuticals only able to provide symptomatic relief rather than treat underlying causes, there is a critical need for nutraceuticals (foods that can treat or prevent disease) like fermented dairy as complementary therapeutic interventions [6, 9].
- García-Burgos M, Moreno-Fernández J, Alférez MJ, Díaz-Castro J, López-Aliaga I. New perspectives in fermented dairy products and their health relevance. Journal of Functional Foods. 2020 Sep 1;72:104059.
- Kim B, Hong VM, Yang J, Hyun H, Im JJ, Hwang J, Yoon S, Kim JE. A review of fermented foods with beneficial effects on brain and cognitive function. Preventive Nutrition and Food Science. 2016 Dec;21(4):297.
- Eroğlu FE, Sanlier N. Effect of fermented foods on some neurological diseases, microbiota, behaviors: mini review. Critical Reviews in Food Science and Nutrition. 2022 Mar 14:1-7.
- Ano Y, Nakayama H. Preventive effects of dairy products on dementia and the underlying mechanisms. International Journal of Molecular Sciences. 2018 Jun 30;19(7):1927.
- Baruah R, Ray M, Halami PM. Preventive and therapeutic aspects of fermented foods. Journal of Applied Microbiology. 2022 May;132(5):3476-89.
- Kumar MR, Azizi NF, Yeap SK, Abdullah JO, Khalid M, Omar AR, Osman M, Leow AT, Mortadza SA, Alitheen NB. Clinical and preclinical studies of fermented foods and their effects on Alzheimer’s disease. Antioxidants. 2022 May;11(5):883.
- Dimidi E, Cox SR, Rossi M, Whelan K. Fermented foods: definitions and characteristics, impact on the gut microbiota and effects on gastrointestinal health and disease. Nutrients. 2019 Aug 5;11(8):1806.
- Vijay A, Valdes AM. Role of the gut microbiome in chronic diseases: A narrative review. European Journal of Clinical Nutrition. 2022 Apr;76(4):489-501.
- Varesi A, Pierella E, Romeo M, Piccini GB, Alfano C, Bjørklund G, Oppong A, Ricevuti G, Esposito C, Chirumbolo S, Pascale A. The potential role of gut microbiota in Alzheimer’s disease: from diagnosis to treatment. Nutrients. 2022 Feb 5;14(3):668.
- Romanenko M, Kholin V, Koliada A, Vaiserman A. Nutrition, gut microbiota, and Alzheimer’s disease. Frontiers in Psychiatry. 2021:1325.
- Sochocka M, Donskow-Łysoniewska K, Diniz BS, Kurpas D, Brzozowska E, Leszek J. The gut microbiome alterations and inflammation-driven pathogenesis of Alzheimer’s disease—a critical review. Molecular Neurobiology. 2019 Mar;56(3):1841-51.
Breast Milk Samples May Inform Early Cancer Diagnosis
- Developments in the analysis of the vesicles in breast milk have identified constituents that are linked to some breast cancers.
- The analysis of breast milk for the purposes of diagnosing breast cancer risk is especially important because the risk of aggressive breast cancer rises after giving birth.
- To speed research progress, investigators argue for a breast milk repository, where unpasteurized human milk is stored for scientific research.
For many years, researchers have thought about the potential for breast milk samples to help in the diagnosis of breast cancers. The idea is practical in the sense that sampling milk is cheap and non-invasive. There are shortcomings to the idea, of course, most obviously that not all women lactate, and those who do lactate do so for a limited period of time. Moreover, the potential for breast milk to offer clues in the diagnosis of breast cancers hinges on scientists developing a sufficiently deep understanding of its components, and of their variation due to factors other than cancer. But this is where research is developing apace. Of particular note are developments in the study of the vesicle contents of breast milk. Yet for the field to really move forward, some researchers argue that a more systematic means of collecting and assessing breast milk would be hugely helpful .
Although using breast milk as a cancer diagnostic may seem like a big step, breast milk samples are already used in the study of disease, and common differences have been established in the milks of women with and without markers of disease. Specifically for metabolic afflictions during pregnancy, such as gestational hypothyroidism and gestational diabetes, researchers have found footprints—associations—of the constituents of mothers’ milk and these medical problems. For example, fewer metabolic and cell-structure proteins have been reported, alongside rising levels of immune proteins, in the colostrum of women with gestational hypothyroidism, a condition where the thyroid gland becomes insufficiently active during pregnancy . These associations serve to make the point that breast milk’s components offer clues as to the state of the maternal body.
There are a few reasons why the analysis of breast milk vesicles might offer potential diagnostics for breast cancers. For various types of cancer, scientists are exploring the idea of using vesicles in body fluids, including saliva, blood and urine, for diagnosis, prognosis and surveillance . But breast milk is especially promising in this regard, as it contains high quantities of these vesicles compared with blood and urine. Moreover, some of the proteins found in breast milk exosomes are known to have oncogenic effects. These include proteins involved in breast involution—the process of remodelling the breast tissue after birth and breastfeeding towards its pre-pregnancy form—that are also linked to metastasis of breast cancers. The proteins in question are matrix metalloproteinase proteins, such as matrix metalloproteinase 2 (MMP-2), MMP-3 and MMP-9.
Such findings go to the heart of why assessing breast cancer risk among women who recently gave birth matters so much. No matter at what age a woman gives birth, having a baby confers a temporarily increased risk of developing breast cancer for a decade after, and indeed for another decade beyond that for women over 35 . During the period half to a full decade after giving birth, the risk of developing metastatic cancer is notably higher . The mechanistic reasons for these elevated risks are frequently linked to processes that happen during postpartum breast involution. Thus, being able to identify in breast milk levels of particular molecules that indicate the degree of elevated risk could select some mothers for more regular check-ups, and lead to much earlier diagnoses—with the potential to save lives. It should be noted that lactating in and of itself reduces the risk of developing breast cancer.
Some research teams claim that the science is mature enough for the use of breast milk in early detection of breast cancers. A review published in May 2022 written by three members of proteomics bioanalytics department at Nestle research in Lausanne, Switzerland, noted that “proteomic tools and methodologies have reached certain maturity” whereby “they are now better suited to discover innovative and robust biofluid biomarkers” . These authors argue that pilot data already supports the hypothesis that specific proteins linked to the early detection and accurate assessment of breast cancer risk can be determined using breast milk samples.
As always, however, more research is needed, and probably a more concerted effort to facilitate it. This is where some form of breast milk repository could be helpful, argue Jeanne Murphy of the U.S. National Cancer Institute, in Bethesda, Maryland, and colleagues . They have in mind a repository similar to the National Children’s Study Vanguard, which is a regional effort. The idea would be to store samples of breast milk from diverse women of different ages, socioeconomic and ethnic groups, at different timepoints in lactation—without processing their milk in such ways as to destroy the chemical components inside, as pasteurization typically does. The authors note that a model project, even though it is for the storage of breast tissue and blood samples rather than milk, is the Komen Tissue Bank. An analogous project to this, storing breast milk samples, would be a long-term investment in the study of “normal” breast milk constituents, and make possible studies using very large numbers of women to assess the performance of breast milk diagnostics.
1. Murphy J., Sherman M. E., Browne E. P., Caballero A. I., Punska E. C., Pfeiffer R. M., Yang H, P., Lee M., Yang H., Gierach G. L. & Arcaro K. F. Potential of Breastmilk Analysis to Inform Early Events in Breast Carcinogenesis: Rationale and Considerations. Breast Cancer Res Treat. 157(1): 13–22. doi:10.1007/s10549-016-3796-x (2016)
2. Dayon L., Cominetti O. & Affolter M. Proteomics of Human Biological Fluids for Biomarker Discoveries: Technical Advances and Recent Applications. Expert Rev Proteomics. (In press, 2022)
3. Xu R., Rai A., Chen M., Suwakulsiri W., Greening D. W. & Simpson R.J. Extracellular vesicles in cancer — implications for future improvements in cancer care. Nat Rev Clin Oncol. 15: 617–638. https://doi.org/10.1038/s41571-018-0036-9 (2018)
4. Borges V. F., Lyons T. R., Germain D. & Schedin P. Postpartum Involution and Cancer: An Opportunity for Targeted Breast Cancer Prevention and Treatments? Cancer Res. 80 (9): 1790–1798. https://doi.org/10.1158/0008-5472.CAN-19-3448 (2020)